Constitutively active fragments of eukaryotic heat shock RNA

ABSTRACT

The present invention provides a novel RNA, designated herein as the “HSR1” (Heat Shock RNA), constitutively active HSR1 fragments, and the use of HSR1 and constitutively active HSR1 fragments for generation of novel therapeutics for the treatment of various diseases in animals and for generation of stress-resistant plants.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application Ser. No. 60/984,614, filed Nov. 1, 2007,and is a continuation-in-part of U.S. patent application Ser. No.11/612,156, filed Dec. 18, 2006, now U.S. Pat. No. 7,919,603, whichclaims priority under 35 U.S.C. 119(e) to U.S. Provisional PatentApplication Ser. No. 60/752,136, filed Dec. 19, 2005, all of which arehereby incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research leading to the present invention was supported, in part, bythe Edward Mallinckrodt Jr. Foundation and grant from NIH GM69800.Accordingly, the U.S. government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention provides a novel RNA, designated herein as the“HSR1” (Heat Shock RNA), and its use together with translationelongation factor eEF1A in activation of heat shock transcription factorHSF. The invention further provides the use of HSR1 for the treatment ofvarious diseases in animals and for generation of stress-resistantplants.

BACKGROUND OF THE INVENTION

All organisms respond to extreme environmental conditions by eitherinducing de novo or dramatically increasing the expression of a numberof genes that protect the cell from the deleterious effect ofintracellular protein denaturation. These genes encode for a family ofproteins called HSPs (heat shock proteins) and other molecularchaperones and cytoprotective proteins. Expression of HSPs and otherchaperones is induced upon exposure to a variety of stressors includingelevated temperature, oxidative stress, alcohol, hyper- and hypoosmoticstress, transition metals, infections, amino acid analogs, etc.(Morimoto, et al., In The Biology of Heat Shock Proteins and MolecularChaperones, 1994 (New York: Cold Spring Harbor Press), pp. 417-455).HSPs and other chaperones are involved in basic cellular processes underboth stress and normal conditions such as correct folding of nascentpolypeptides, binding to exposed hydrophobic regions of denatured orabnormal proteins to prevent their aggregation and promote degradationor assembly, and translocation of proteins into membrane-boundorganelles in the cell (see, e.g., Ellis, Trends Biochem Sci., 2000, 25:210-212; Forreiter and Nover, J. Biosci., 1998, 23: 287-302; Hartl andHayer-Hartl, Science, 2002, 295: 1852-1858; Haslbeck, Cell Mol. Life.Sci., 2002, 59: 1649-1657; Young et al, Trends Biochem. Sci., 2003, 28:541-547; Soll and Schleiff, Nature Rev. Mol. Cell. Biol., 2004, 5:198-208). Expression of some HSPs is essential during embryogenesis(Luft, et al., Chaperones, 1999; 4:162-170). HSPs function primarily bystabilizing partially unfolded states. They do not contain specificinformation for correct folding, but rather prevent unproductiveinteractions (aggregation) between non-native proteins. This type ofmolecular chaperones can be distinguished from other heat-inducedproteins acting as direct folding catalysts like peptidylprolyl-cis/trans isomerases (e.g., cyclophilins, FKBPs) or proteindisulfide isomerases (Schmid, Curr. Biol., 1995, 5: 993-994; Guidebookto molecular chaperones and protein-folding catalysts, Gething (ed.),Oxford Univ. Press, 1997; Gothel and Marhiel, Cell Mol. Life. Sci.,1999, 55: 423-436; He et al., Plant Physiol., 2004, 134: 1248-1267; Tuand Weissman, J. Cell Biol., 2004, 164: 341-346; Kadokura et al., Annu.Rev. Biochem., 2003, 72: 111-135). Four major aspects in the life cycleof proteins invoke chaperone activities (Guidebook to molecularchaperones and protein-folding catalysts, Gething (ed.), Oxford Univ.Press, 1997): (i) they ensure that nascent polypeptides emerging fromthe ribosome are kept in a folding competent state until the wholesequence information is available; (ii) since fully folded proteinscannot be translocated through membranes, chaperones are needed tomaintain or create a partially unfolded form of proteins destined forthe import into mitochondria or plastids (Braun and Schmitz, Planta,1999, 209: 267-274; Neupert and Brunner, Nat. Rev. Mol. Cell. Biol.,2002, 3: 555-565; Rehling et al., J. mol. Biol., 2003, 326: 639-657;Soll and Schleiff, Nature Rev. Mol. Cell. Biol., 2004, 5: 198-208);(iii) they stabilize damaged proteins generated as a result of chemicalor physical stress and thus facilitate renaturation and/or degradationin the recovery period; (iv) they assist and control assembly anddisassembly of multiprotein complexes (Lorimer, Plant Physiol., 2001,125: 38-41; Kim et al., J. Biol. Chem., 2002, 47: 44778-44783). Manydata collected during the last few years indicate that members ofdifferent Hsp families act together in so-called “chaperone machines”(Bukau and Horwich, Cell, 1998, 92: 351-366; Walter and Buchner, Angew.Chem.-Int. Edit., 2002, 41: 1098-1113; Young et al., Trends Biochem.Sci., 2003, 28: 541-547), and different chaperone complexes may interactto generate a network for protein maturation, assembly and targeting(Frydman, Annu. Rev. Biochem., 2001, 70: 603-647; Johnson and Craig,Cell, 1997, 90: 201-204; Forreiter and Nover, J. Biosci., 1998, 23:287-302; Lee and Vierling, Plant Physiol., 2000, 122: 189-198). Althoughmany proteins are potential substrates for chaperone machines, e.g.after stress-induced protein damage, most of them (about 80%) fold in achaperone-independent manner under normal conditions (Netzer and Hartl,Trends Biochem. Sci., 1998, 23: 68-73). It is assumed that proteins atthe surface of the ribosomes may help to stabilize nascent polypeptidechains (Frydman, Annu. Rev. Biochem., 2001, 70: 603-647; Hartl andHayer-Hartl, Science, 2002, 295: 1852-1858).

HSP family proteins are classified into several groups based on theirmolecular weight and sequence homology between bacteria, plants andanimals. Hsp101 (ClpA/B/X in prokaryotes) are involved in ATP-dependentdissociation of protein aggregates. Hsp90 (HtpG in prokaryotes) functionas co-regulators of signal transduction complexes. Under stressconditions, HSP90 binds to exposed hydrophobic regions of denaturedproteins, while in the absence of stress it participates in fundamentalcellular processes such as hormone signaling and cell cycle control(Pearl, et al., Curr. Opin. Struct. Biol. 2000; 10:46-51). Manyregulatory proteins, including steroid hormone receptor, cell cyclekinases, and p53 have been identified as HSP90 substrates (Young, etal., J. Cell Biol. 2001; 154:267-273; Pratt, Annu. Rev. Pharmacol.Toxicol. 1997; 37:297-326). HSP90 is ubiquitously expressed and mayconstitute up to 1-2% of total cellular protein. Mammalian cells expressat least two HSP90 isoforms, HSP90α and HSP90β, which are encoded by twoseparate genes (Pearl, et al., Adv. Protein Chem. 2001; 59:157-186).Hsp70/Hsp40 (DnaK/DnaJ in prokaryotes) are involved in primarystabilization of newly formed proteins via ATP-dependent binding andrelease (Mayer, et al., Adv. Protein Chem. 2001; 59:1-44), unfolding andrefolding of proteins during their transport across membranes (Jensen,et al., Curr. Biol. 1999; 9:R779-R782; Pilon, et al., Cell 1999;97:679-682; Ryan, et al., Adv. Protein Chem. 2001:59:223-242), andbinding to partially denatured, abnormal, or targeted for proteasomedegradation proteins (Zylicz, et al., IUBMB. Life 2001; 51:283-287).HSP70 subfamily includes both constitutive and stress-inducible proteinsthat are closely related and often referred to as Hsc70 and HSP72respectively. HSP40 is a co-chaperon for HSP70 class proteins, whichmodulates ATPase activity and substrate binding properties of the latter(Ohtsuka, et al., Int. J. Hyperthermia 2000; 16:231-245). Hsp60/Hsp10(GroEL/GroES in prokaryotes) function in cytosol and organelles tostabilize unfolded states and assist refolding or degradation in anATP-dependent manner. Hsp20 are a family of small HSPs, which includesprimate HSP27, rodent HSP25, αA-crystallins and αB-crystallins. HSP25/27is expressed constitutively and expression increases after exposure toheat, transition metal salts, drugs, and oxidants. Small HSPs form highmolecular weight oligomeric complexes (e.g., oligomers consisting of8-40 monomers) that serve as binding sites for stabilization of unfoldedproteins until they can be refolded by HSP70/HSP40 and/or Hsp101 system(Van Montfort, et al., Adv. Protein Chem. 2001; 59:105-156; Welsh, etal., Ann. N. Y. Acad. Sci. 1998; 851:28-35).

The induction of HSP gene expression occurs primarily at transcriptionallevel and is mediated by a family of transcription factors named HSF(Heat Shock Factor). Only one HSF has been identified in yeast,Drosophila, and C. elegans, and three HSFs have been identified invertebrates (Wu, Ann. Rev. Cell Dev. Biol. 1995; 11:441-469; Morimoto,Genes and Development, 1998, 12: 3788-3796; Nakai, Cell StressChaperones, 1999, 4:86-86-93; Nover et al., Cell Stress Chaperones,2001, 6: 177-189; recently, a fourth, HSF-like open reading frame (ORF)(HsfY) encoded on the Y chromosome was identified in vertebrates—seeTessari et al., Mol. Human. Reprod., 2004, 10: 253-258). In human cells,three HSFs (HSF1, HSF2, and HSF4) have been characterized (Morimoto, etal., Genes Dev. 1998; 12:3788-3796). HSF1 is ubiquitously expressed andplays the principle role in the stress-induced expression of HSPs. It isan apparent functional analog of Drosophila HSF.

In contrast to few well-defined HSFs in other classes of organisms,plants are characterized by a great complexity of the plant HSF family.The complexity of the plant HSF gene family is thought to allow a highlyflexible and efficient response to rapid changes in environmentalconditions that accompany the stationary lifestyle of plants (Nover etal., Cell Stress Chaperones, 2001, 6: 177-189; Kotak et al., Plant J.,2004, 39: 98-112). Due to their immobility, plants had to adapt to growand propagate under extreme environmental conditions, including variousenvironmental abiotic stresses such as dehydration/extreme waterdeficiency, unfavorable high or low temperatures or abrupt temperatureshifts, high salinity, heavy metal stress, acid rain, high lightintensities, UV-light, grafting, or the bending of shoots or stems inresponse to wind and/or rain, or biotic stresses such as pathogenattack.

The model plant Arabidopsis thaliana contains 21 HSF genes, as well asseveral genes encoding HSF-like proteins (The Arabidopsis GenomeInitiative 2000, Nature, 408: 796-815). More than 16 HSF genes werefound in tomato (Scharf et al., Mol. Cell. Biol., 1998, 18: 2240-2251;Treuter et al., Mol. Gen. Genet., 1993, 240: 113-125; Boscheinen et al.,Mol. Gen. Genet., 1997, 255: 322-331; Bharti et al., Plant J., 2000, 22:355-365; Bharti et al., Plant Cell, 2004, 16: 1521-1535; Doring et al.,Plant Cell, 2000, 12: 265-278; Heerklotz et al., Mol. Cell. Biol., 2001,21: 1759-1768; Mishra et al., Genes Dev., 2002, 16: 1555-1567; Port etal., Plant Physiol., 2004, 135: 1457-1470; see also reviews by Nover etal., Cell Stress Chaperones, 2001, 6: 177-189; Bharti and Nover, Heatstress-induced signalling; in Plant signal transduction: Frontiers inmolecular biology, Scheel and Wastemack eds., Oxford Univ. Press, 2002,pp. 74-115), and many other HSF genes were identified in rice, maize andother species (Goff et al., Science, 2002, 296: 92-100; Yu et al. 2002,Science, 2002, 296: 79-92). The number of plant HSFs continues to grow.More than 60 new class A HSFs were identified from expressed sequencetag (EST) databases, including 19 new HSFs in soybean (34 in total) andat least 23 HSFs in rice (Kotak et al., Plant J., 2004, 39: 98-112).

Plant HSF genes are assigned to three different classes (classes A, Band C) according to their unique structural characteristics (Nover etal., Cell Stress Chaperones, 2001, 6: 177-189). Class A HSF proteinscomprise the largest group of HSFs with 15 proteins in Arabidopsis. Theycontain an activation domain at the C-terminus and are thought to beinvolved in transcriptional activation. Class B and class C HSFs lack adefined aromatic/hydrophobic/acidic (AHA)-type activation domain(reviewed in Miller and Mittler, Annals of Botany, 2006, 98: 279-288).The absence of an activation domain, as well as their inability torescue the yeast HSF1 mutation, has led to the assumption that class BHSFs function as repressors (Boscheinen et al., Mol. Gen. Genetics,1997, 255: 322-331; Czarnecka-Vemer et al., Plant Mol. Biol., 2000, 43:459-471, Czarnecka-Vemer et al., Plant Mol. Biol., 2004, 56: 57-75).However, HSFB1 was recently demonstrated to function as a novelco-regulator of the tomato HSFA1 or HSFA2 enhancing theirtranscriptional activity (Bharti et al., Plant Cell, 2004, 16:1521-1535). In tomato (Lycopersicon peruvianum), (i) HSFA1a is themaster regulator responsible for heat stress (hs)-induced geneexpression including synthesis of HSFA2 and HSFB1. It is indispensablefor the development of thermotolerance. (ii) Although functionallyequivalent to HSFA1a, HSFA2 is exclusively found after heat shockinduction and represents the dominant HSF, the “working horse” of theheat shock response in plants subjected to repeated cycles of heat shockand recovery in a hot summer period. Tomato HSFA2 is tightly integratedinto a network of interacting proteins (HSFA1a, Hsp17-CII, Hsp17-CI)influencing its activity and intracellular distribution. (iii) Becauseof structural peculiarities, HSFB1 acts as co-regulator enhancing theactivity of HSFA1a and/or HSFA2. But in addition, it cooperates with yetto be identified other transcription factors in maintaining and/orrestoring housekeeping gene expression. (reviewed in Baniwal et al., J.Biosci., 2004, 29: 471-487)

The situation in Arabidopsis seems to be different in several aspects. Asingle HSF as master regulator could not be identified (Lohmann et al.,Mol. Gen. Genomics, 2004, 271: 11-21). In addition, Arabidopsis HSFB1 isnot comparable to its tomato counterpart (Kotak et al., Plant J., 2004,39: 98-112).

Based on the analysis of Arabidopsis HSFs, it is suggested that there isa high degree of specialization in the response of specific HSFs toparticular stress conditions. Thus, for example, AtHSFA9 appears to bespecific to salt, drought and cold stress, while AtHSFA6a and AtHSFA6bappear to be cold and salt specific. With the exception of AtHSFA2 andAtHSFB1, the pattern of HSF expression during heat stress is differentfrom the pattern of HSF expression during other stresses. Because HSEsare found in the promoters of many defense genes (see, e.g., Rizhsky etal., J. Biol. Chem., 2004, 279: 11736-11743), it is possible thatdifferent HSFs, expressed during different stresses, activate or controldifferent defense pathways. The combinatorial function of HSFs couldtherefore be responsible for stress-specific expression of HSPs or otherdefense genes, and specific stress conditions could therefore causeactivation of a particular set(s) of different HSFs (Rizhsky et al.,Plant Physiol., 2004, 134: 1683-1696). Thus, in addition to beingpotentially redundant, the HSF gene network in plants is highly flexibleand specialized. It controls the response of plants to diverse stressconditions, as well as potentially their combination (Rizhsky et al.,Plant Physiol., 2004, 134: 1683-1696; Mittler, Trends in Plant Sci.,2006, 11: 15-19). Indeed, functional interdependence studies betweenHSFs, co-immunoprecipitation and yeast one-hybrid assays suggest thatall class A HSFs of tomato can interact with each other, potentiallyforming hetero-oligomers (Scharf et al., Mol. Cell. Biol., 1998, 18:2240-2251; Bharti et al., Plant J., 2000, 22: 355-365). Furthermore,different HSFs can associate with each other potentially functioning asco-activators or co-repressors (Baniwal et al., J. Biosciences, 2004,29: 471-487). Taken together, the complexity of the HSF gene network ofplants is evident on at least five different levels: (1) a large numberof HSF genes are present in the plant genome; (2) each HSF gene canpotentially bind to its own promoter, as well as to the promoters of allother HSF genes; (3) monomers encoded by different HSF genes caninteract leading to activation or suppression of transcription; (4)monomers encoded by different HSF genes can interact affecting nucleartargeting and retention; and (5) spatial and temporal expressionpatterns of HSFs could affect different responses in different tissues.These features make the HSF gene network a highly redundant andspecialized network that functions in a stress- ordevelopmental-specific manner (reviewed in Miller and Mittler, Annals ofBotany, 2006, 98: 279-288).

Under normal conditions, mammalian HSF1 exists in the cell as aninactive monomer. Following exposure to elevated temperature, HSF1trimerizes and apparently relocates to the nucleus where it binds tospecific sites in HSP promoters upstream of the transcription initiationsite (Wu, Ann. Rev. Cell Dev. Biol. 1995; 11:441-469; Westwood, et al.,Mol. Cell. Biol. 1993; 13:3481-3486; Westwood, et al., Nature 1991;353:822-827). Similarly, plant HSF1A undergoes trimerization and DNAbinding upon stress (Mishra et al., Genes Dev, 2002, 16: 1555-1567). TheHSF binding site contains arrays of inverted repeats of the elementNGAAN designated HSE (Heat Shock Element). The same evolutionarilyconserved HSE sequence is recognized by all members of the HSF proteinfamily and is universal to all eukaryotic species (Kim et al., ProteinSci. 1994; 3:1040-1051). The heat shock promoter is primed for rapidactivation in response to heat shock. Many factors of the basaltranscription machinery are bound to the promoter prior to heat shockincluding GAGA factor, TFIID, transcriptionally arrested RNA polymeraseII located 21-35 nucleotides downstream of the transcription start site,and presumably some other transcription factors (Shopland, et al., GenesDev. 1995; 9:2756-2769). The partitioning of HSF molecules between thenucleus and cytoplasm is a subject to some controversy since, in thecase of Xenopus laevis, HSF1 was shown to be a nuclear protein beforeheat shock (Mercier, et al., J. Biol. Chem. 1997; 272:14147-14151),while in most studies employing mammalian cells, HSF1 was found in boththe cytoplasm and nucleus under normal conditions (Sarge, et al., Mol.Cell. Biol. 1993b; 13:1392-1407). Interestingly, heat shock treatment ofHeLa cells results in rapid and reversible localization of HSF1 inspecific nuclear granules, which constitute a novel type of nuclearprotein compartmentalization (Cotto, et al., Journal of Cell Science1997b; 110:2925-2934). The granules appear within 30 sec of heat shocktreatment and rapidly disappear upon shift to normal temperature.However, the functional significance of this phenomenon is stillunknown.

The overall structure of HSF1 is conserved among species as distant asDrosophila and human. The DNA-binding domain is just over 100 aminoacids long and is situated close to the N-terminus of the molecule. Thisdomain is about 70% homologous between human HSF1 and Drosophila HSF andshows 55% homology between human HSF1 and yeast HSF. The leucine zipperdomain, which is C-terminal with respect to the DNA-binding domain, iseven more conserved showing 79% homology between human and Drosophila.In vertebrates, this domain comprises three hydrophobic heptad repeatswith an additional heptad repeat located in the C-terminus of HSF1. Ithas been implicated in the maintenance of the inactive monomeric stateof HSF1 under non-stressful conditions (Wu, et al., In The Biology ofHeat Shock Proteins and Molecular Chaperones, 1994 (New York: ColdSpring Harbor Press), pp. 395-416). It has been suggested that thefunction of HSFs as transcription activators resides in short activatorpeptide motifs (AHA motifs) in their C-terminal domains characterized byaromatic (W, F, Y), large hydrophobic (L, I, V) and acidic (E, D) aminoacid residues (Treuter et al., Mol. Gen. Genet., 1993, 240: 113-125;Döring et al., Plant Cell, 2000, 12: 265-278; Kotak et al., Plant J.,2004, 39: 98-112). Similar AHA motifs were found and functionallycharacterized in the centre of many other transcription factors of yeastand mammals, e.g. VP16, RelA, Spl, Fos, Jun, Gal4, Gcn4 as well as thesteroid and retinoic acid receptors (see summary and references inDöring et al., Plant Cell, 2000, 12: 265-278; Kotak et al., Plant J.,2004, 39: 98-112).

A number of models have been proposed to explain how HSF activation isregulated, most of them focusing on repression of the inactive monomerunder normal conditions as the most probable mode of regulation. Severallines of evidence suggest the existence of a titratable cellular factorthat acts to repress HSF under normal conditions by keeping it in amonomeric form. Indeed, overexpression of both HSF1 and HSF2 in 3T3mouse fibroblasts resulted in constitutive activation of theirDNA-binding activity and transcription of HSP genes (Sarge, et al., Mol.Cell. Biol. 1993a; 13:1392-1407). The observed effect could reflecteither general cellular stress caused by the drastic increase in HSFconcentration or titration of the negative regulator of HSF, which ispresent in limiting amounts. Furthermore, expression of human HSF1 inDrosophila cells results in a decrease of the activation thresholdtemperature by 9 degrees, to the temperature characteristic for the heatshock conditions in Drosophila (32° C.) instead of 41° C.—acharacteristic threshold for mammalian cells. At the same time,Drosophila HSF expressed in human cells remained constitutively activeeven when the temperature was lowered to 25° C.—the normal growthtemperature for Drosophila (Clos, et al., Nature 1993; 364:252-255).Similarly, Arabidopsis HSF remained active in Drosophila and human cellseven under control conditions (Hubel, et al., Mol. Gen. Genet. 1995;248:136-141). Taken together, these results strongly suggest that theintracellular environment rather than structure of the HSF moleculedetermines its behavior in response to heat stress. These data areconsistent with the possibility that HSF activation is mediated by aspecific stimulating factor(s).

The process of HSF1 activation can be divided into at least twosteps: 1) trimerization and acquisition of DNA binding activity; 2)acquisition of transactivation competence, which is correlated withhyperphosphorylation of the factor. Treatment with salicylate and othernon-steroid anti-inflammatory drugs induces HSF trimerization and DNAbinding but fails to stimulate transcription of HSP genes (Jurivich, etal., J. Biol. Chem. 1995a; 270:24489-24495). However, majority of HSFregulation occurs at the level of its trimerization.

The model of HSF regulation, where HSF activity is a subject to thenegative feedback mechanism involving inducible HSP72 and otherchaperones, has been a paradigm for a decade. According to this model,HSF monomer is present in the complex with HSP72 and other chaperones(most notably HSP90) under normal conditions. Trimerization of HSFmolecules is thought to occur spontaneously as soon as negativeregulation by HSPs has been relieved. Indeed, in a number of studies HSFhas been shown to possess intrinsic responsiveness to heat (Zuo, et al.,Mol. Cell. Biol. 1995; 15:4319-4330; Farkas, et al., Molecular andCellular Biology 1998a; 18:906-918). However, the HSF concentrationsused in these studies far exceeded those found in the cell, whichquestions the physiological relevance of the data. Furthermore, althoughall these studies imply that HSF trimerization occurs spontaneously oncethe negative regulation is relieved, the existence of a positiveregulation of HSF activity can not be ruled out. For example, the rapid,specific and reversible formation of HSF granules in nuclei during heatshock (Cotto, et al., Journal of Cell Science 1997a; 110:2925-2934;Jolly, et al., Journal of Cell Science 1997; 110:2935-2941) testifiesagainst spontaneous mechanism given the relatively low number of HSFmolecules in the cell, their even distribution throughout cytoplasmunder normal conditions, and molecular crowding effect due to very hightotal protein concentration in the cell as compared to in vitroexperimental systems.

HSPs, and HSP70 family in particular, is considered a part of aprotective mechanism against certain pathological conditions, includingischemic damage, infection, and inflammation (Pockley, Circulation 2002;105:1012-1017). In the case of inflammation, a protective role of HSPshas been shown in a variety of experimental models (Jattela et al., EMBOJ. 1992; 11:3507-3512; Morris et al., Int. Biochem. Cell Biol. 1995;27:109-122; Ianaro et al., FEBS Lett. 2001; 499:239-244; Van Molle etal., Immunity 2002; 16:685-695). For example, Ianaro et al. (Mol.Pharmacol. 2003; 64:85-93) have recently demonstrated that HSF1-induced(see below) HSP72 expression in the inflamed tissues and activation ofthe heat shock response is closely associated with the remission of theinflammatory reaction. It follows, that HSP genes may function asanti-inflammatory or “therapeutic” genes, and their selective in vivotransactivation may lead to remission of the inflammatory reaction(Ianaro et al., FEBS Lett. 2001; 499:239-244 and Ianaro et al., FEBSLett. 2001; 508:61-66).

A potential therapeutic value of causing increased expression of HSPs inindividuals suffering from cerebral or cardiac ischemia andneurodegenerative diseases has been also suggested (Klettner, Drug NewsPerspect. 2004; 17:299-306; Hargitai et al., Biochem. Biophys. Res.Commun. 2003; 307:689-695; Yenari et al., Ann. Neurol. 1998; 44:584-591;Suzuki et al., J. Mol. Cell. Cardiol. 1998; 6:1129-1136; Warrik et al.,Nat. Genet. 1999; 23:425-428). For example, Zou et al. (Circulation2003; 108:3024-3030) have recently shown that cardiomyocyte cell deathinduced by hydrogen peroxide was prevented by overexpression of HSF1 inCOS7 cells. Thermal preconditioning at 42° C. for 60 minutes activatedHSF1, which played a critical role in survival of cardiomyocytes fromoxidative stress. Ischemia/reperfusion injury has been reported toinduce apoptosis in cardiomyocytes (Fliss and Gattinger, Circulation1996; 79:949-956). Zou et al. (Circulation 2003; 108:3024-3030) havealso demonstrated that, in the heart of transgenic mice overexpressing aconstitutively active form of HSF1 (and having elevated levels of HSPs27, 70 and 90 in the heart), ischemia followed by reperfusion-inducedST-segment elevation in ECG was recovered faster, infarct size wassmaller, and cardiomyocyte death was less than in wild-type mice. Theseresults suggest that elevated activity of HSF1 (and levels of HSPs)exert protective effect on the electrical activity of myocardium againstischemia/reperfusion injury (see also Plumier et al., J. Clin. Invest.1995; 95:1854-1860; Marber et al., ibid., pp. 1446-1456; Radford et al.,Proc. Natl. Acad. Sci. USA, 1996; 93:2339-2342).

HSPs and HSF1 have been also implicated in protection against a varietyof neurodegenerative disorders that involve aberrant protein folding andprotein damage. Neuronal cells are particularly vulnerable in this senseas HSF activity and HSP expression are relatively weak in such cells andmotor neurons appear to require input of HSP secreted from adjacentglial cells to maintain adequate molecular chaperone levels (Batulan etal., J. Neuosci. 2003; 23:5789-5798; Guzhova et al., Brain Res. 2001;914:66-73).

Polyglutamine (polyQ) expansion is a major cause of inheritedneurodegenerative diseases called polyglutamine diseases. Several polyQdiseases have been identified, including Huntington's disease (HD),spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy,Kennedy disease, and five forms of spinocerebellar ataxia. Aggregates orinclusion bodies of polyQ proteins (e.g., huntingtin) within thenucleus, or in the cytoplasm of neuronal cells in some Huntington'sdisease patients, are a prominent pathological hallmark of most polyQdiseases (Davies et al., Cell 1997; 90:537-548; DiFiglia et al., Science1997; 277:1990-1993). Formation of polyQ protein inclusions correlateswith an increased susceptibility to cell death (Warrik et al., Cell1998; 94:939-49). The early stages of aggregates are highly toxic tocells (Bucciantini et al., Nature 2002; 416:507-11). Suppression ofaggregates should be beneficial to cells and could delay the progressionof polyQ diseases (Sanchez et al., Nature 2003; 421:373-9). HSPs havebeen implicated in many of these neurodegenerative diseases based on theassociation of chaperones with intracellular aggregates. For example,live cell imaging experiments show that Hsp70 associates transientlywith huntingtin aggregates, with association-dissociation propertiesidentical to chaperone interactions with unfolded polypeptides (Kim etal., Nat. Cell Biol. 2002; 4:826-31). This suggests that these chaperoneinteractions may reflect the efforts of Hsp70 to direct the unfoldingand dissociation of substrates from the aggregate. Moreover,overexpression of the Hsp70 chaperone network suppresses aggregateformation and/or cellular toxicity. A critical protective role for smallHSPs (HSP27) has been also recently demonstrated in Huntington's disease(Wyttenbach et al., Human Mol. Gen. 2002; 11:1137-51). Collectively,these observations have led to the hypothesis that the elevated levelsof heat shock proteins reduce or dampen aggregate formation and cellulardegeneration (Warrick et al, Nat. Genet. 1999; 23:425-8; Krobitsch andLindquist, Proc. Natl. Acad. Sci. USA 2000; 97:1589-94). Importantly,HSF1 overexpression suppressed polyQ-inclusion formation even betterthan any combination of HSPs in culture cells and in Huntington'sdisease model mice extending their life span (Fujimoto et al., J. Biol.Chem. 2005; 280:34908-16).

Multiple HSPs are also overexpressed in brains from Alzheimer's (AD) andParkinson's disease (PD) patients and found to be associated with senileplaques and Lewy bodies, respectively. These HSPs may be involved inneuroprotection by various mechanisms ranging from microglia activationand clearance of amyloid-β peptides to suppression of apoptosis(Kitamura and Nomura, Pharmacol Ther. 2003; 97:35-33).

Aging is also associated with the decrease in activity of HSF (Tonkisand Calderlwood, Int. J. Hyperthermia 2005; 21:433-444). Indeed,neurodegenerative diseases often occur later in life when heat shockgenes seem to be induced poorly (Soti and Csermely, Exp. Gerontol. 2003;38:1037-40; Shamovsky and Gershon, Mech. Ageing Dev. 2004; 125:767-75).Moreover, it has been recently shown that induction of heat shock eitherby temperature or HSF overexpression could extend life span in modelorganisms. For example, the heat shock response has recently beenimplicated in the regulation of longevity in C. elegans in a pathwaythat overlaps with the insulin signaling pathway (Hsu et al., Science2003; 300:1142-5; Morley and Morimoto, Mol. Biol. Cell 2004; 15:657-64).Reduction of HSF1 levels cause a decreased life span in C. elegans,similar to life span effects observed in mutants of Daf-16, a FOXOtranscription factor in the insulin signaling pathway. Daf-16 and HSF1share a subset of downstream target genes, including small HSPs. RNAinterference experiments showed that a decrease in small HSPs and otherHSPs leads to a decrease in longevity (Hsu et al., Science 2003;300:1142-5; Morley and Morimoto, Mol. Biol. Cell 2004; 15:657-64).Similarly, Walker et al. (Aging Cell 2003; 2:131) have demonstrated thatoverexpression of HSP16 can extend C. elegans' life span. Therefore, inaddition to the prevention of diseases of aging, increased levels ofHSPs may lead to increases in life span (Westerheide and Morimoto, J.Biol. Chem. 2005, 280:33097-100).

Heat shock is a known transcriptional activator of humanimmunodeficiency virus type 1 (HIV) long terminal repeat (LTR). However,HIV LTR suppression can occur under hyperthermic conditions.Specifically, suppression of the HIV LTR was observed in a conditionalexpression system for gene therapy applications that utilizes theheat-inducible promoter of the human heat shock protein 70B gene toenhance the HIV LTR-driven therapeutic gene expression afterhyperthermia (temperature higher than 37° C.) (Gerner et al., Int. J.Hyperthermia 2000; 16:171-181). Similarly, the inhibition of HIVtranscription has been reported after whole-body hyperthermia at 42° C.in persons with AIDS (Steinhart et al., J. AIDS Hum. Retrovirol. 1996;11:271-281). Recently demonstrated ability of a mutant transcriptionallyactive HSF1 (lacking C-terminal residues 203-315) to suppress HIVpromoter activity further suggests that HSF1 could serve as a tool forthe treatment of AIDS (Ignatenko and Gerner, Exp. Cell Res. 2003;288:1-8; see also Brenner and Wainberg, Expert Opin. Biol. Ther. 2001;1:67-77).

Due to interaction of HSPs with numerous regulatory proteins (e.g.,NF-κB, p53, v-Src, Raf1, Akt, steroid hormone receptors) and pathways(e.g., inhibition of c-Jun NH2-terminal kinase (JNK) activation,prevention of cytochrome c release, regulation of the apoptosome,prevention of lysosomal membrane permeabilization, prevention of caspaseactivation) involved in the control of cell growth, the increasedproduction of HSPs has potent anti-apoptotic effect (Bold, et al.,Surgical Oncology-Oxford 1997; 6:133-142; Jaattela, et al., Exp. CellRes. 1999; 248:30-43; Nylandsted, et al., Ann. N. Y. Acad. Sci. 2000;926:122-125; Pratt and Toft, Exp. Biol. Med. (Maywood) 2003; 228:111-33;Mosser and Morimoto, Oncogene 2004; 23:2907-18). Anti-apoptotic andcytoprotective activities of HSPs directly implicate them in oncogenesis(Jolly and Morimoto, J. Natl. Cancer Inst. 2000; 92:1564-72; Westerheideand Morimoto, J. Biol. Chem. 2005, 280:33097-100). Many cancer cellsdisplay deregulated expression of HSPs, whose elevated levels contributeto the resistance of cancerous cells to chemo- and radiotherapy.Different subfamilies of HSPs including HSP70, HSP90, and HSP27 werefound to be expressed at abnormal levels in various human tumors(Cardoso, et al., Ann. Oncol. 2001; 12:615-620; Kiang, et al., Mol. CellBiochem. 2000; 204:169-178). In some cases, HSPs are expressed at cellsurface in tumors, most probably serving as antigen presenting moleculesin this case (Conroy, et al., Eur. J. Cancer 1998; 34:942-943). BothHSP70 and HSP90 were demonstrated to mediate cytoplasmic sequestrationof p53 in cancer cells (Elledge, et al., Cancer Res. 1994;54:3752-3757). Inactivation of wild-type p53 function has been observedin variety of cancer cells and is in fact one of the most commonhallmarks in human cancer (Malkin, et al., J. Neurooncol. 2001;51:231-243). Other studies have demonstrated that HSP70 has a potentgeneral antiapoptotic effect protecting cells from heat shock, tumornecrosis factor, serum starvation, oxidative stress, chemotherapeuticagents (e.g., gemcitabine, torootecan, actinomycin-D, campothecin, andetoposide), and radiation (Jaattela, et al., EMBO J. 1992; 11:3507-3512;Jaattela, et al., J. Exp. Med. 1993; 177:231-236; Simon, et al., J.Clin. Invest 1995; 95:926-933; Karlseder, et al., Biochem. Biophys. Res.Commun. 1996; 220:153-159; Samali and Cotter, Exp. Cell Res. 1996;223:163-170; Sliutz et al., Br. J. Cancer 1996; 74:172-177). At the sametime, HSP70 is abundantly expressed in human malignant tumors of variousorigins, not only enhancing spontaneous growth of tumors, but alsorendering them resistant to host defense mechanisms and therapeutictreatment (Ciocca, et al., Cancer Res. 1992; 52:3648-3654). Therefore,finding a way to suppress HSP overproduction in cancerous cells will beinvaluable for increasing the efficacy of the existing anti-cancertherapeutic approaches.

HSF1-mediated induction of HSPs has been also implicated in protectionof sensory hair cells against acoustic overexposure, hyperthermia andototoxic drugs. It has been shown that mice lacking HSF1 have reducedrecovery from noise-induced hearing loss (Altschuler et al., AudiolNeotol. 2003; 7:152-156). Similarly, Sugahara et al. (Hear Res. 2003;182:88-96) have demonstrated that the loss of sensory hair cells wasmore significant in HSF1-null mice compared with that of wild-type micewhen mice were subjected to acoustic overexposure. They have also shownthat the loss of both the sensory hair cells and the auditory functioninduced by acoustic overexposure was inhibited by pretreatment of theinner ear with local heat shock.

Developing stress tolerant plants has been a growing agriculturalconcern as will help farmers across the world to cope with ever-presentenvironmental stresses and with increased stresses due to climate changeand will also help to achieve higher yields. However, traditional plantbreeding strategies to develop new lines of plants that exhibitresistance (tolerance) to various types of biotic and abiotic stressesare relatively slow and require specific resistant lines for crossingwith the desired line. Limited germplasm resources for stress toleranceand incompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to stress tolerance (e.g.,drought, cold, and salt tolerance) in plants are complex in nature andinvolve multiple mechanisms of cellular adaptation and numerousmetabolic pathways. This multi-component nature of stress tolerance hasnot only made breeding for stress tolerance largely unsuccessful, buthas also limited the ability to genetically engineer stress tolerantplants using biotechnological methods. Thus, generation of geneticallyengineered plants where multiple stress-response pathways are affectedappears an attractive alternative approach.

Based on the information provided above, HSF appears to be an attractivetherapeutic target for regulating HSP synthesis to combat variousdiseases in animals and to generate stress-resistant plants (Mestril etal., J. Clin. Invest. 1994; 93:759-67; Morimoto, et al., Genes Dev.1998; 12:3788-3796; Jolly and Morimoto, J. Natl. Cancer Inst. 2000;92:1564-72; Ianaro et al., FEBS Lett. 2001; 499:239-44; Calderwood andAsea, Int. J. Hyperthermia 2002; 18:597-608; Zou et al., Circulation2003; 108:3024-30; Westerheide and Morimoto, J. Biol. Chem. 2005;280:33097-100).

SUMMARY OF THE INVENTION

As follows from the Background Section, there is a clear need in the artto develop novel therapeutically effective regulators of HSF. Thepresent invention satisfies this and other needs by disclosing for thefirst time two HSF-associated factors, translation elongation factoreEF1A and a novel Heat Shock RNA 1 (HSR1), that mediate HSF activationupon stress under physiological conditions. Among the two HSF-associatedfactors, HSR1 serves as a cellular thermosensor that determines thetemperature threshold for the heat shock response. As disclosed herein,eEF1A and HSR1 form a ternary ribonucleotprotein complex with HSF1 invitro and in vivo. The present invention further provides noveltherapeutics that affect activity of the ternary complex or any of itsindividual components and in this way can be used to treat variousdiseases in animals (e.g., cancer, inflammation, ischemia,neurodegeneration, age-related diseases, HIV infection, deafness, andrelated disorders) and to generate stress-resistant plants.

Specifically, the first object of the present invention is to provide anisolated ribonucleotide molecule comprising a eukaryotic Heat Shock RNA1 (HSR1) or a fragment thereof. In one embodiment, the present inventionprovides an isolated ribonucleotide molecule comprising hamster HSR1cloned from BHK cells having SEQ ID NO: 1. In another embodiment, thepresent invention provides an isolated ribonucleotide moleculecomprising human HSR1 cloned from HeLa cells having SEQ ID NO: 37. In aseparate embodiment, the invention provides a 3′-truncatedconstitutively active fragment of human HSR1 (HSR1-435) having SEQ IDNO: 35. In yet another embodiment, the present invention provides anisolated ribonucleotide molecule comprising HSR1 of Arabidopsis havingSEQ ID NO: 34. In yet another embodiment, the present invention providesan isolated ribonucleotide molecule comprising Drosophila HSR1 havingSEQ ID NO: 33. In yet another embodiment, the present invention providesan isolated ribonucleotide molecule comprising C. elegans HSR1 havingSEQ ID NO: 36.

In a separate embodiment, the invention also provides an isolatedpolynucleotide molecule (e.g., a gene or vector) encoding a eukaryoticHSR1 or a fragment thereof. Thus, in a specific embodiment, theinvention provides a human genomic sequence (SEQ ID NO: 32) comprisingthe sequence encoding human HSR1 and unique flanking sequences andflanking inverted repeats. In another embodiment, the invention providesan Arabidopsis genomic sequence (SEQ ID NO: 31) comprising the sequenceencoding Arabidopsis HSR1 with flanking inverted repeats.

The invention also provides an isolated single-stranded polynucleotidemolecule comprising a nucleotide sequence that is the complement of anucleotide sequence of one strand of any of the aforementionednucleotide sequences. In a specific embodiment, the present inventionprovides aHSR1 ribonucleotide molecule comprising a nucleotide sequence(SEQ ID NO: 3) that is the complement of hamster HSR1 having SEQ ID NO:1.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that is homologous to thenucleotide sequence of the HSR1 or the HSR1-encoding polynucleotidemolecule of the invention or fragments thereof (including, among others,constitutively active HSR1 fragments homologous to human HSR1-435). In aspecific embodiment, such polynucleotide molecule has at least 50%sequence identity, preferably at least 75% sequence identity, morepreferably at least 90% sequence identity, and most preferably at least95% sequence identity to at least 100 consecutive nucleotides of thenucleotide sequence of the HSR1 or the HSR1-encoding polynucleotidemolecule of the invention. Particularly useful HSR1 orthologs of thepresent invention are human, hamster, mouse, Xenopus, Drosophila, C.elegans, and yeast orthologs as well as various plant orthologsincluding, without limitation, Arabidopsis and commercially importantplants such as sugar storing and/or starch-storing plants, for instancecereal species (rye, barley, oat, wheat, maize, millet, sago etc.),rice, pea, marrow pea, cassava, sugar cane, sugar beet, potato, tomato,cow pea or arrowroot, fiber-forming plants (e.g. flax, hemp, cotton),oil-storing plants (e.g. rape, sunflower), protein-storing plants (e.g.legumes, cereals, soybeans), vegetable plants (e.g. lettuce, chicory,Brassicaceae species such as cabbage), fruit trees, palms and othertrees or wooden plants being of economical value such as in forestry,forage plants (e.g. forage and pasture grasses, such as alfalfa, clover,ryegrass), and ornamental plants (e.g. roses, tulips, hyacinths,camellias or shrubs). In addition to the sequence homology, thepreferred HSR1 orthologs of the present invention possess at least oneof the same functional properties of the hamster HSR1 or human HSR1.Such properties include without limitation the ability to interact witheEF1A, the ability to interact with HSF, the ability to form a ternarycomplex with eEF1A and HSF, the ability to activate HSF DNA binding, theability to undergo a conformational change in response to stress, etc.As further provided herein, HSR1 orthologs are constitutively expressedin vivo and are functionally interchangeable.

The present invention further provides isolated polynucleotide andoligonucleotide molecules that hybridize to the HSR1 or theHSR1-encoding polynucleotide molecules of the invention under standardor high stringency hybridization conditions. The invention also providesseveral specific non-limiting examples of such polynucleotide andoligonucleotide molecules, including without limitation oligonucleotidemolecules having SEQ ID NOS: 4-6, 9-12, and 17-19.

The HSR1-related nucleic acid molecules of the invention or the nucleicacid molecules comprising sequences that hybridize to them understandard hybridization conditions (including their homologs/orthologs,complementary sequences and various oligonucleotide probes and primersderived from them) can be used to modulate (e.g., inhibit or augment) afunction of HSR1 or HSF/HSR1/eEF1A ternary complex (e.g., by modulating(i) interaction between HSR1 and eEF1A, (ii) interaction between HSR1and HSF, (iii) formation of a ternary complex HSF/HSR1/eEF1A, (iv)activation of HSF DNA binding, (v) the ability of HSR1 to undergo aconformational change in response to stress, etc.). The HSR1-relatednucleic acid molecules of the invention or the nucleic acid moleculescomprising sequences that hybridize to them under standard hybridizationconditions (including their homologs/orthologs, complementary sequencesand various oligonucleotide probes and primers derived from them) can bealso used to modulate (e.g., inhibit or activate) expression of HSR1genes (e.g., by inhibiting transcription, processing, transport, or bypromoting degradation of corresponding RNAs). In a specific embodiment,the present invention provides HSR1-specific antisense oligonucleotides,RNA interference (RNAi) molecules, ribozymes, and triple helix formingoligonucleotides (TFOs) which can be effectively used to mediate any ofthese functions. The invention also provides specific non-limitingexamples of HSR1-specific antisense oligonucleotides including withoutlimitation molecules having nucleotide sequences selected from the groupconsisting of SEQ ID NO: 3 (aHSR1), SEQ ID NO: 9 (1^(HSR1)), SEQ ID NO:10 (2^(HSR1)), SEQ ID NO: 11 (5^(HSR1)), and SEQ ID NO: 12 (6^(HSR1)).The invention also provides specific non-limiting examples ofHSR1-specific RNAi molecules including without limitation siRNAmolecules comprising SEQ ID NO: 7 (siHSR1-160) or SEQ ID NO: 20(siHSR1-224).

In a related embodiment, the present invention provides recombinantvectors and host cells (both eukaryotic and prokaryotic) which have beengenetically modified to express or overexpress various nucleotidemolecules of the present invention. In a specific embodiment, theinvention provides a eukaryotic cell which has been genetically modifiedto express or overexpress a constitutively active HSR1 molecule (e.g., a3′-truncated human HSR missing 100-150 3′ nucleotides such as humanHSR1-435 or its ortholog in other species). In a preferred embodiment,such cell as a result of such expression of a constitutively active HSR1molecule has an increased stress resistance. In one embodiment, suchcell is a mammalian cell. In another embodiment, such cell is a plantcell. The invention also provides genetically modified animals (e.g.,mammals) or plants that comprise such cells.

The present invention also provides a eukaryotic cell that has beengenetically modified so that its normal expression of an HSR1-encodinggene has been reduced or eliminated. In another embodiment, theinvention provides a eukaryotic cell that has been genetically modifiedso that its normal expression of an HSR1-encoding gene has beenincreased. In a preferred embodiment, such cell as a result of suchincreased expression of HSR1-encoding gene has an increased stressresistance. In a preferred embodiment, such cells are mammalian cells orplant cells. The invention also provides genetically modified animals(e.g., mammals) or plants that comprise such cells.

In conjunction with the HSR1-specific antisense oligonucleotides, RNAimolecules, ribozymes, and TFOs, the present invention also provides amethod for modulating (e.g., inhibiting or increasing) a stress responsein a cell comprising administering said molecules to the cell. Asspecified herein, the modulation of the stress response in a cell can bedetectable by various methods, including without limitation (i)detecting changes in the level of HSF1-mediated transcription and (ii)detecting changes in the level of a Heat Shock Protein (HSP). In arelated embodiment, the invention provides a method of modulating (e.g.,inhibiting or increasing) a stress tolerance in a cell comprisingadministering to the cell an antisense oligonucleotide or an RNAimolecule or a ribozyme or a TFO of the invention.

Another object of the present invention is to use the HSR1-specificantisense oligonucleotides, RNAi molecules, ribozymes, and TFOs of theinvention as a basis for developing therapeutics to treat variousdiseases (e.g., cancer, inflammation, ischemia, reperfusion injury,neurodegenerative disorders, age-related diseases, HIV infection,deafness, and related disorders).

Accordingly, the present invention provides novel anti-cancer agentsbased on the HSR1-specific antisense oligonucleotides, RNAi molecules,ribozymes, and TFOs as well as methods for using such agents to treatcancer. The novel anti-cancer agents of the present invention can beused in conjunction with existing treatments to improve their effect byincreasing the sensitivity of the cells to pro-apoptotic stimuli such asthermo-, chemo-, and radiotherapeutic treatments.

Thus, in one embodiment, the present invention provides a method forincreasing sensitivity of a cancer cell to a treatment comprisingadministering to the cell an antisense oligonucleotide or an RNAimolecule or a ribozyme or a TFO that can specifically inhibit a functionof an HSR1 or that can specifically inhibit expression of a geneencoding an HSR1. In a specific embodiment, the treatment can beselected from the group consisting of (without limitation) radiationtreatment, chemical treatment, thermal treatment, and any combinationthereof. In another embodiment, the invention provides a method fortreating a cancer in a mammal comprising administering to the mammal anantisense oligonucleotide or an RNAi molecule or a ribozyme or a TFOthat can specifically inhibit a function of an HSR1 of the invention orthat can specifically inhibit expression of a gene encoding an HSR1 ofthe invention. In yet another embodiment, the invention provides amethod for improving efficiency of an anti-cancer treatment in a mammalcomprising administering to the mammal an antisense oligonucleotide oran RNAi molecule or a ribozyme or a TFO that can specifically inhibit afunction of an HSR1 of the invention or that can specifically inhibitexpression of a gene encoding an HSR1 of the invention. In a specificembodiment, the mammal is human. In another specific embodiment, themammal is further subjected to a treatment selected (without limitation)from the group consisting of radiation therapy, chemotherapy,thermotherapy, and any combination thereof. The relative timing ofantisense/RNAi/ribozyme/TFO administration and anti-cancer treatmentwould depend on the delivery mechanism for antisense/RNAi/ribozyme/TFOand on the type of the specific anti-cancer treatment used. Generally,cells may become more sensitive to an anti-cancer treatment as soon asone hour after antisense/RNAi/ribozyme/TFO administration.

The invention also provides a method for inhibiting/treating a diseasein a mammal comprising administering to the mammal an antisenseoligonucleotide or an RNAi molecule or a ribozyme or a TFO that canspecifically augment a function of an HSR1 of the invention or that canspecifically activate expression of a gene encoding an HSR1 of theinvention. The diseases that can be inhibited/treated using such methodinclude without limitation an inflammatory reaction, ischemia,reperfusion injury, HIV transcription, aging, age-related diseases,neurodegeneration (e.g., Alzheimer's disease, Parkinson's disease,amyotrophic lateral sclerosis (ALS), Huntington's disease, spinobulbarmuscular atrophy, dentatorubral pallidoluysian atrophy, spinocerebellarataxias, Kennedy disease, etc.), and deafness. In a specific embodiment,the mammal is human.

Another object of the present invention is a generation ofstress-resistant plants (i) by generation of plants which aregenetically modified to express or overexpress a constitutively activeHSR1 molecule (e.g., a 3′-truncated human HSR missing 100-150 3′nucleotides such as human HSR1-435 or its plant ortholog) or (ii) bygeneration of plants which are genetically modified so that their normalexpression of an HSR1-encoding gene has been increased or (iii) byadministering to such plants an antisense oligonucleotide or an RNAimolecule or a ribozyme or a TFO that can specifically augment a functionof an endogenous HSR1 or that can specifically activate expression of agene encoding an endogenous HSR1. The resistance to stresses achieved insuch plants includes without limitation various environmental abioticstresses such as dehydration/extreme water deficiency, unfavorable highor low temperatures or abrupt temperature shifts, high salinity, heavymetal stress, acid rain, high light intensities, UV-light, grafting, orthe bending of shoots or stems in response to wind and/or rain, orbiotic stresses such as pathogen attack.

In another object, the present invention provides a method foridentifying a candidate compound useful for modulating a function of anHSR1 of the invention and/or HSF/HSR1/eEF1A ternary complex, said methodcomprising:

(a) contacting a first cell with a test compound for a time periodsufficient to allow the cell to respond to said contact with the testcompound;

(b) determining in the cell prepared in step (a) the function of theHSR1 and/or HSF/HSR1/eEF1A ternary complex; and

(c) comparing the function of the HSR1 determined in step (b) to thefunction of the HSR1 and/or HSF/HSR1/eEF1A ternary complex in a second(control) cell that has not been contacted with the test compound;

wherein a detectable change in the HSR1 and/or HSF/HSR1/eEF1A ternarycomplex in the first cell in response to contact with the test compoundcompared to the function of the HSR1 and/or HSF/HSR1/eEF1A ternarycomplex in the second cell that has not been contacted with the testcompound, indicates that the test compound modulates the function of theHSR1 and/or HSF/HSR1/eEF1A ternary complex and is a candidate compound.

As disclosed herein, a function of HSR1 and/or HSF/HSR1/eEF1A ternarycomplex assayed according to this method can be any function, e.g.,stress/temperature-induced conformational change of HSR1, interaction ofHSR1 with HSF, interaction of HSR1 with eEF1A, formation ofHSF/HSR1/eEF1A ternary complex, activation of HSF-mediated DNA binding,activation of HSP expression, thermotolerance, etc. The test compoundcan be without limitation a small inorganic molecule, a small organicmolecule, a polypeptide, a nucleic acid molecule, or a chimera orderivative thereof. In one embodiment of this screening method, bothtest and control cells can be subjected to stress (e.g., heat shock). Inthis embodiment, the test compound can be added after cells had beensubjected to stress, or after a preconditioning stress but before thelethal stress, or before cells had been subjected to stress.

In a separate embodiment, the present invention provides a method foridentifying a candidate compound capable of binding to the HSR1 of theinvention and/or HSF/HSR1/eEF1A ternary complex, said method comprising:

(a) contacting the HSR1 and/or HSF/HSR1/eEF1A ternary complex with atest compound under conditions that permit binding of the test compoundto the HSR1 and/or HSF/HSR1/eEF1A ternary complex; and

(b) detecting binding of the test compound to the HSR1 and/orHSF/HSR1/eEF1A ternary complex.

The test compound can be without limitation a small inorganic molecule,a small organic molecule, a polypeptide, a nucleic acid molecule, or achimera or derivative thereof. The binding of the test compound to theHSR1 and/or HSF1/HSR1/eEF1A ternary complex can be detected, e.g., bydetecting HSF DNA binding in crude extracts, HSP expression, or cellthermotolerance. In a specific embodiment, the conditions that permitbinding of the test compound to the HSR1 and/or to the HSF1/HSR1/eEF1Aternary complex are stress conditions (e.g., heat shock conditions).

The above-identified screening methods of the invention can be used toidentify a candidate compound that can be used to treat a condition thatcan be treated by modulating a function of a eukaryotic HSR1 and/orHSF/HSR1/eEF1A ternary complex. Such conditions include withoutlimitation cancer, inflammation, ischemia, reperfusion injury,neurodegeneration, age-related diseases, HIV infection, deafness, andrelated disorders. The above-identified screening methods of theinvention can be also used to identify a candidate compound that can beused to generate stress-resistant plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B represent SDS-PAGE analysis of the proteins from the lysateof heat shocked BHK-21 (A) or HeLa (B) cells that interact withbacterially expressed GST-HSF1 immobilized on glutathione Sepharose. A.Lane 1: the whole lysate of heat shocked BHK cells; lane 2: supernatantafter the incubation of the lysate of heat shocked BHK cells with HSF1Sepharose beads; lane 3: proteins bound to HSF1 Sepharose after theincubation with the lysate of heat shocked BHK cells and washing; lane4: proteins bound to HSF1 Sepharose following three successive rounds ofelution at 43° C.; lanes 5-7: proteins released from HSF1 Sepharose inthree successive rounds of elution at 43° C. B. Lane 1: supernatantafter the incubation of the lysate of heat shocked HeLa cells with HSF1Sepharose beads; lane 2: proteins bound to HSF1 Sepharose after theincubation with the lysate of heat shocked HeLa cells and washing; lane3: proteins bound to HSF1 Sepharose following three successive rounds ofelution at 43° C.; lanes 4-6: proteins released from HSF1 Sepharose inthree successive rounds of elution at 43° C. The analysis reveals apolypeptide of about 45 kDa which was retained on HSF1 Sepharose afterincubation with the lysate of heat shocked BHK-21 or HeLa cells but notfrom the lysate of unstressed cells. FIG. 1C represents Western blottinganalysis of purified recombinant HSF used in pull-down experiments(using anti-HSF1 monoclonal antibody from Stresgene Co.) showing thatHSF1 coupled to Sepharose was predominantly in monomeric form (withnegligibly low amount of trimers). Where indicated, cross-linking wasperformed using 0.5 mM EGS.

FIGS. 2A-C represent electrophoretic gel mobility shift assays (EMSA) ofin vitro induction of HSF1 DNA binding by the HSF1-interacting fractionfrom the lysate of heat shocked BHK cells. A. EMSA of the whole celllysate of unstressed BHK cells (20 μg) incubated in the absence (lane 1,5) or in the presence (lanes 2-4) of fractions 5-7 from FIG. 1A. “43°”in lane 1 indicates that the lysate was heat-shocked for 15 min prior toEMSA. Lanes 6-9 contain fraction 5 from FIG. 1A. “HSE” (lanes 6-7)indicates the inclusion of an excess of unlabeled HSE oligonucleotide.“Ab” (lanes 8-9) indicates the presence of a monoclonal anti-HSF1antibody that causes a supershift (lane 9). This EMSA demonstrates thatthe eEF1A-containing fraction from the lysate of heat shocked BHK cellsactivates endogenous HSF1 in a lysate from unstressed BHK cells. Asshown in lanes 6-9, this activity is specific. B. EMSA of the increasingamounts of purified recombinant mouse HSF1 incubated in the absence (−)or presence of the eEF1A-containing fraction from the lysate of heatshocked BHK cells isolated on HSF1-Sepharose (see FIG. 1). C. EMSAshowing a dose-dependent activation of HSF1 in a lysate of unstressedBHK cells after 20 μg of a lysate of unstressed (C) or heat shocked (HS)cells were incubated with increasing amounts of the eEF1A-containingfraction (1-10 μl). EMSA in B and C demonstrate that theeEF1A-containing fraction from the lysate of heat shocked BHK cellsinduces DNA binding activity of HSF1, and this effect is dose-dependentwith respect to both the amount of pure HSF1 (B) or cell lysate (C) andthe eEF1A-containing fraction.

FIG. 2D represents Western blotting analysis of purified recombinantmouse HSF1 incubated in the absence (−) or presence of theeEF1A-containing fraction, followed by cross-linking with EGS. Afterseparation of proteins by SDS-PAGE and transfer to nitrocellulosemembrane, the blot was probed with an anti-HSF1 8487 polyclonalantibody. The immunoblotting demonstrates that the eEF1A-containingfraction induces trimerization of purified recombinant HSF1.

FIG. 3A represents Western blotting analysis of co-immunoprecipitationof eEF1A and HSF1 demonstrating formation of an eEF1A-HSF1 complex invivo. BHK cells were heat shocked for the indicated periods of time,whole cell lysates were prepared and eEF1A was immunoprecipitated withanti-eEF1A antibody, followed by SDS-PAGE and immunoblotting withanti-HSF1 8487 polyclonal antibody. 0-60 min: duration of the heat shocktreatment at 43° C.; 60R: 60 min heat shock followed by recovery for 60min at 37° C.

FIG. 3B represents electrophoretic gel mobility shift assays (EMSA)showing the effect of cytochalasin on HSF1 activation by HS. HeLa cellswere incubated with 10 μM cytochalasin (“Cyt”; lanes 3, 4) and eithersubjected to 1 h HS at 43° C. (lanes 2, 3) or maintained at 37° C.(lanes 1, 4). Whole cell lysates were then prepared and assayed by EMSA.The EMSA demonstrates that, while cytochalasin does not affect HSF1binding to DNA in the absence of HS (lane 4), its addition 1 h prior toexposure of cells to HS results in a dramatic increase in HSF activity(lanes 2 and 3).

FIG. 4A represents electrophoretic gel mobility shift assays (EMSA) ofin vitro induction of HSF1 DNA binding in a whole cell lysate (10 μgtotal protein) from heat shocked (HS, 1 h at 43° C.) BHK cells treatedwith RNase A (500 ng; for 1 h at 37° C.) in the presence of variousconcentrations of tRNA (ng). The amount of HSF1 in each lane wasmonitored by immunoblotting with anti-HSF1 antibodies (lower panel). Theassay shows dependence of the HSF1 activation on RNA. Control lanes 1and 2 show HSF1 EMSA in an extract from HS cells before and afterincubation for 1 h at 37° C., respectively.

FIG. 4B represents denaturing 4% PAGE analysis of RNA (HSR1) isolatedfrom the pooled HSF1-interacting (eEF1A containing) fractions from thelysate of heat shocked HeLa or BHK cells. Where indicated HSR1 sampleswere treated for 30 min at 25° C. with DNase I (10 U) or RNase A (100ng) before loading onto the gel (right lanes). The gel was silverstained. The analysis shows that HSF1-interacting RNA (HSR1) migratingat about 2.5 kb on denaturing PAGE is sensitive to RNase A but not toDNase I.

FIG. 4C represents electrophoretic gel mobility shift assays (EMSA) ofin vitro induction of HSF1 DNA binding by the whole cell lysate (20 μg)of heat shocked BHK cells which was first incubated with micrococcalnuclease (MNase), then, after reaction was stopped by addition of EGTA(lane 1), it was incubated with RNA (either HSR1 (lane 2) or total RNA(lane 3)) for 1 hour at room temperature. Lane 4: lysate of unstressedcells. The analysis demonstrates that HSR1 restored the DNA bindingactivity of HSF1 after it has been eliminated using micrococcalnuclease.

FIG. 4D represents Northern blot analysis of HSR1 expression in BHK andHeLa cells. Total RNA (10 μg) from either heat shocked (HS) orunstressed (C) BHK or HeLa cells was subjected to electrophoresis in adenaturing agarose gel, transferred to a membrane, and probed with[³²P]-labeled RNA probe corresponding to region 167-405 of HSR1. An 18SRNA probe was used for normalization purposes.

FIG. 5A demonstrates reconstitution of the HSF1 activating complex.Schematics on the top show HSR1 cDNA flanked with T7 and T3 promoters.The lower panel represents electrophoretic gel mobility shift assays(EMSA) of recombinant HSF1 incubated with pure eEF1A and HSR1 isolatedeither from HeLa or BHK activating fractions (lanes 7, 8) or eithersense (T3) or antisense (T7) HSR1 synthesized by in vitro transcription(lanes 9-11). Quantitation of HSF1 activation is presented as the foldincrease relative to a background control (lane 1). The analysis showsthat both HSR1 from heat-shocked BHK or HeLa cells (lanes 7, 8) andsense in vitro synthesized HSR1-T3 (lane 10) activate HSF1 (lanes 7, 9,and 10: ˜5% of the total HSF1 activated under these conditions) whenadded together with purified eEF1A, while neither component alone iscapable of activating HSF1 (lanes 2-6).

FIG. 5B represents Western blotting analysis of HSF1 oligomerizationduring activation in vitro. Purified recombinant mouse HSF1 wasincubated with eEF1A with (lanes 2-4) or without (lane 1) HSR1-T3 in thepresence (lane 2) or absence (lanes 1, 3, 4) of RNase A, followed bycross-linking with indicated concentrations of EGS, SDS-PAGE, andimmunoblotting using HSF1 8487 polyclonal antibody. The analysis showsthat HSR1-T3-mediated HSF1 activation is accompanied by HSF1trimerization.

FIG. 5C represents electrophoretic mobility analysis of endogenous HSR1isolated from BHK cells or HSR1 synthesized by in vitro transcription.HSR1 isolated from BHK cells (lanes 3, 4, and 6) or synthesized by invitro transcription (T3, lanes 1, 2, and 5) was incubated in Mg²⁺-freebuffer (lanes 1 and 3) or in buffer containing 4 mM Mg²⁺ (lanes 2, 4-6)and subjected to electrophoresis in an 1% agarose gel. Samples in lanes5 and 6 were heated at 43° C. prior to loading. M-RNA ladder.

FIG. 5D represents agarose gel analysis of RT-PCR products after HSR1co-immunoprecipitation with an anti-eEF1A antibody (performed asdescribed in the legend to FIG. 3A), followed by isolation of RNA from aprecipitate prepared from HS (lane 3) or control (lane 4) cells andRT-PCR using HSR1-specific primers. Lane 1—no reverse transcriptionstep. Lane 2—in vitro transcribed HSR1 (T3) was used in place of HSR1isolated from the immunoprecipitate. The analysis demonstrates theformation of a molecular complex comprising HSR1 and eEF1A, which ismore abundant in cells subjected to stress.

FIG. 5E represents mapping of the functional domains of HSR1 byelectrophoretic gel mobility shift assays (EMSA). 15 overlapping 45-merHSR1 antisense DNA oligos covering the entire length of HSR1 werescreened for their ability to inhibit HSF1 activation in thereconstituted system. Recombinant HSF1 (<10 nM) was activated in thepresence of pure eEF1A (0.01 mg/ml) and HSR1-T3 (0.4 μM). Whereindicated, an antisense oligo was added to 10 μM. Changes in HSF1activation were quantitated after setting the activity of HSF1 in theabsence of oligo at 100% (lane 2) and without eEF1A at 0% (lane 1). Notethat some oligos potentiate HSF1 activation (e.g., oligo #10). Theanalysis demonstrates that at least two domains in HSR1 (defined byoligos 1-2 and 5-6, respectively) are essential for HSF1 activation.

FIGS. 6A-E demonstrate the effect of HSR1-specific antisenseoligonucleotides/siRNA transfected into BHK or HeLa cells on heat shockresponse in vivo. A. Electrophoretic gel mobility shift assays (EMSA) ofHSF1 DNA binding activity observed in the lysate of BHK cells after heatshock at 43° C. for 1 h in the presence of 6^(HSR1) HSR1 antisenseoligonucleotide or various controls. BHK cells were transfected with 100nM of either 6^(HSR1) antisense oligonucleotide (lane 2), an equalamount of the corresponding double-stranded oligo(6^(HSR1)/anti-6^(HSR1), lane 3) or mock transfected (lane 1).Transfected cells were heat-shocked for 1 h at 43° C., a whole celllysate was immediately prepared from half of the cells and 20 μg of thetotal protein was analyzed by EMSA for HSF1 activity. The other half ofthe transfected cells was allowed to recover at 37° C. for 16 h and awhole cell lysate was analyzed by immunoblotting using anti-HSP72(inducible HSP70) antibody (lower panel). Control (lane 5)—lysate ofunstressed cells. HS (lane 4)—lysate of untransfected heat shockedcells. Upper panel shows quantitation of EMSA data from 3 independentexperiments. The analysis demonstrates that 6^(HSR1), but not6^(HSR1)/anti-6^(HSR1), inhibits activation of HSF1 in vivo as well asthe production of HSP72 in response to HS. B. Graph of BHK cellviability. Cells were transfected with 100 nM 6^(HSR1) or anti-6^(HSR1),or mock transfected. Thermotolerance was induced by incubation for 1 hat 43° C. followed by 15 h recovery at 37° C. Lethal HS (45° C.) wasperformed for the indicated time periods. Cell viability was determinedby MTS assay 16 h after lethal HS. The data for 6^(HSR1) andanti-6^(HSR1) oligos were normalized to the efficiency of transfection.The data show that 6^(HSR1), but not 6^(HSR1)/anti-6^(HSR1), compromisescell survival after HS. C. Graph showing inhibition of HS promoteractivation by HSR1-directed siRNA. RLuc activity was measured in lysatesof BHK cells transiently transfected with a mixture of RLuc reporterplasmid, a β-gal internal control, and a siRNA construct expressingHSR1-directed siRNA (siHSR1) corresponding to the 6^(HSR1) antisenseoligo or its mutant derivative (siHSR1: C→G). C—control lysate ofunstressed cells. The data demonstrate that, while the RLuc activity wasinduced about 200-fold by HS treatment followed by recovery at 37° C.,siHSR1 strongly inhibited the HS induction of RLuc. D. Graph showinginhibition of HS response in stably transfected cells expressingHSR1-directed siRNA (siHSR1). RLuc activity was measured in lysates ofHeLa cells stably expressing GFP (HS), siHSR1 or antisense HSR1 (aHSR1)transiently transfected with a RLuc reporter plasmid and a β-galinternal control. C—lysate of unstressed cells. The HSF1 activity in thelysates of GFP (HS) and siHSR1 expressing cells is shown in the inset.The data demonstrate that cells expressing siHSR1 or aHSR1 (but not GFP)were deficient in their ability to induce RLuc activity after 2 h HS at43° C. followed by overnight recovery at 37° C. E. Graph showinginhibition of thermotolerance by HSR1-directed siRNA (siHSR1). Cellsstably expressing GFP or siHSR1 were subjected to lethal 2 h HS at 46°C. after being pre-conditioned by 1 h 43° C. HS where indicated. Cellviability was determined by the MTS-based assay. The data demonstratethat cells stably expressing siHSR1 failed to acquire thermotoleranceafter HS pre-conditioning. Taking together, the data in A-E show thatHSR1 is essential for the HS response in mammalian cells.

FIGS. 7A-B are schematic representations of generation of derivatizedHSR1 by “walking” with E. coli RNA polymerase without (A) or with (B)“roadblock”. “Ni-NTA” stands for Ni-agarose beads.

FIG. 8 shows a BLAST b12seq sequence comparison of Drosophila (SEQ IDNO: 33) and human (SEQ ID NO: 37) HSR1. The sequence comparison reveals98% identity with only 8 substitutions (shown by asterisks).

FIGS. 9A-C represent the results of electrophoresis mobility shiftassays (EMSA) of HSF1 DNA binding activity to radiolabled DNA fragmentcontaining heat shock element (HSE). A. shows EMSA analysis of HSF1activation in vitro by 3′-truncated HSR1-435 deletion mutant (SEQ ID NO:35) as compared to the full-length human HSR1. HSF1 activation reactionswere set up with 0.1, 1 and 10 nM HSR1-435 and HSR1 in each triplet andwere incubated for 15 min at 23° C. or 43° C. followed by the additionof HSF1 and eEF1A. As shown in the panel, HSF1 is activated in thepresence of HSR1-435 regardless of the incubation temperature in adose-dependent manner. In contrast, in the presence of the full lengthHSR1, the activation of HSF1 only occurs when the RNA is pre-exposed toelevated temperature (43° C.). Thus, in contrast to the full-lengthhuman HSR1, 3′-truncated human HSR1-435 deletion mutant (SEQ ID NO: 35)is constitutively active. B. shows EMSA analysis of the effect of DTT onHSF1 activation. HSF1 activation reactions were set up either in theabsence (empty circles) or in the presence (filled circles) of DTT (2mM) and HSR1 (1 nM). The reactions were then incubated at 37° C. or at43° C. (as indicated below the gel) for 15 min. As shown in the panel,HSF1 activation does not occur in the absence of DTT. In the presence of2 mM DTT, HSF1 is activated if HSR1 is pre-incubated at 43° C. prior tothe addition of HSF1 and eEF1A. It follows that DTT is required for theactivation of HSF1 in vitro by eEF1A and HSR1 indicating the involvementof Cys residues in this process. C. shows EMSA analysis of HSF1activation by HSR1 isolated from human and Drosophila used to assess theextent of the functional conservation of the key components of the HSFactivating complex. Left three lanes: HSF1 alone, HSF1 and HSR1, HSF1and eEF1A. Next five lanes: 1 nM human HSR1 incubated at the followingtemperatures for 15 min: 23.1° C., 30.8° C., 36.4° C., 39° C., 43.9° C.before the addition of HSF1 and eEF1A. Last nine lanes: 1 nM DrosophilaHSR1 incubated at the following temperatures for 15 min: 22.9° C., 24.1°C., 25.9° C., 28.2° C., 30.8° C., 33.6° C., 36.4° C., 39° C., 41.2° C.before the addition of HSF1 and eEF1A. As shown in the panel, human HSR1starts to activate HSF1 at approximately 36° C. and the activationreaches the maximum at approximately 43° C., consistent with themammalian heat shock threshold temperature in vivo. Drosophila HSR1starts to activate HSF1 at approximately 30° C. and the maximum ofactivation is reached at approximately 36° C. As follows from thesedata, the threshold temperature of HSF1 activation in in vitro system iscontrolled by HSR1 making HSR1 a bona fide thermosensor. The resultsalso demonstrate that Drosophila HSR1 and human HSR1 are interchangeablein their ability to activate HSF1 at temperatures corresponding toDrosophila and human heat shock thresholds, respectively.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Within the meaning of the present invention, when used in relation to acell of a eukaryotic organism, the terms “stress” or “stressfulconditions” refer to any condition that results in activation orincrease of Heat Shock Factor (HSF)-mediated transcription or inactivation or increase of the synthesis of at least one heat shockprotein (HSP). Examples of stressful conditions as applied to animalsinclude without limitation elevated temperature, oxidative stress (e.g.H₂O₂), alcohols, hyper- and hypoosmotic stress, heavy metals, amino acidanalogs, viral infection, inflammation, and serum starvation (see alsoMorimoto, et al., In The Biology of Heat Shock Proteins and MolecularChaperones, 1994 (New York: Cold Spring Harbor Press), pp. 417-455).Examples of stressful conditions as applied to plants include withoutlimitation various environmental abiotic stresses such asdehydration/extreme water deficiency, unfavorable high or lowtemperatures or abrupt temperature shifts, high salinity, heavy metalstress, acid rain, high light intensities, UV-light, grafting, or thebending of shoots or stems in response to wind and/or rain, or bioticstresses such as pathogen attack (see, e.g., WO 2005/113777 or WO2005/033318).

The terms “heat shock” or “HS” or “heat stress” is used to refer tostressful conditions associated with elevated temperature. Examples ofheat shock conditions for different eukaryotic organisms include withoutlimitation 40-43° C. for mammalian cells, 29-36° C. for Drosophila cellsand Xenopus (normal growth 25° C.), 28° C. for salmon and trout, 40-45°C. for Arabidopsis and corn plants, and 39-40° C. for yeast.

The term “thermotolerance” is used herein to refer to a cellularadaptation caused by a single, severe but nonlethal heat exposure thatallows a cell or organism to survive a subsequent and otherwise lethalheat stress.

In connection with the present invention, the term “stress resistantplant” or “stress resistance of plants” refers to the property of agiven plant or plant species to protect itself against either abioticstress or biotic stress. Preferably, “stress resistance” refers to meanswith which a plant reacts to abiotic or biotic stress. This means thatthe provisions of the invention preferably improve or establishresistance against stress by modifying the stress response of a plant inthat it reacts either quicker or slower to stress than a plant being notmodified according to the methods for improving stress resistance ofplants which are described herein.

Said term thus also refers to a significant reduction of susceptibilityto a pathogen in plants treated according to the provisions of thepresent invention as compared to corresponding untreated plants. Theterm “susceptibility” refers to the capacity of a given pathogen to growon or in the tissue of a plant. In particular, “susceptibility” refersto the growth of a pathogen on the epidermal surface and from there intothe epidermis and subepidermal tissue, e.g. the mesophyll. Thus, theterm “susceptibility” also covers incidents of pathogen attacks wherethe pathogen grows for a certain while on the host plant, however,without being capable to take up nutrients from the host and thereforewithout successfully colonizing the host plant. In particular,successful colonization is characterized by completing that part of thepathogen's life cycle which takes place on the plant host. With regardto fungal pathogens, like for instance powdery mildew, such a successfulcolonization is for instance apparent from the formation of a haustoriumand of secondary hyphae. In the case of an attack of Cladosporim fulvumsuccessful colonization is for instance apparent from having entered thehost plant and having begun the sexual life cycle. In particular, areduction of susceptibility may be evident from a significant reductionof penetration events and/or a significant reduction of hypersensitivereactions as for instance visible by fluorescence detection. Preferably,such a reduction of susceptibility of a plant so-treated is by at least10%, more preferably at least 20%, still more preferably by at least50%, even more preferably by at least 80% and most preferably toapproximately 100% as compared to an untreated plant and with respect tothe number of penetration events and/or hypersensitive reactions. Thelevel of resistance is measured using conventional methods. For example,the level of resistance to a pathogen may be determined by comparingphysical features and characteristics (for example, plant height andweight, or by comparing disease symptoms, for example, delayed lesiondevelopment, reduced lesion size, leaf wilting and curling, water-soakedspots, amount of pathogen growth, and discoloration of cells) of thenon-naturally occurring plant (e.g., a transgenic plant).

In the context of the present invention, the term “biotic stress” meansany form of biotic stress for a plant, including without limitationstress caused by attack or settlement or colonization of a plant byplant pathogens, such as fungi, bacteria, protozoa, insects, nematodes,viruses, or viroids.

“Abiotic stress” is any form of stress for a plant, including withoutlimitation stress caused by dehydration/extreme water deficiency,unfavorable high or low temperatures or abrupt temperature shifts, highsalinity, heavy metal stress, acid rain, high light intensities,UV-light, ozone, irradiation, grafting, bending of shoots or stems inresponse to wind, rain, or disease, or mechanical wounding caused bychewing insects.

The terms “heat shock protein” or “HSP” are used interchangeably torefer to a family of proteins involved in basic cellular processes underboth stress and normal conditions such as correct folding of nascentpolypeptides, binding to exposed hydrophobic regions of denatured orabnormal proteins to prevent their aggregation and promote degradationor assembly, and translocation of proteins into membrane-boundorganelles in the cell (see, e.g., Ellis, Trends Biochem Sci., 2000, 25:210-212; Forreiter and Nover, J. Biosci., 1998, 23: 287-302; Hartl andHayer-Hartl, Science, 2002, 295: 1852-1858; Haslbeck, Cell Mol. Life.Sci., 2002, 59: 1649-1657; Young et al, Trends Biochem. Sci., 2003, 28:541-547; Soll and Schleiff, Nature Rev. Mol. Cell. Biol., 2004, 5:198-208; Mayer, et al., Adv. Protein Chem. 2001; 59:1-44; Jensen, etal., Curr. Biol. 1999; 9:R779-R782; Pilon, et al., Cell 1999;97:679-682; Ryan, et al., Adv. Protein Chem. 2001; 59:223-242; Zylicz,et al., IUBMB. Life 2001; 51:283-287). HSPs are classified into severalgroups based on their molecular weight and include without limitationHsp101, HSP90, Hsp70 (e.g., Hsc70 and Hsp72), Hsp40, Hsp60, Hsp20 (e.g.,HSP27, HSP25, α-crystallins), and Hsp10.

As used herein, the terms “heat shock factor” or “HSF” refer to a familyof transcription factors which are involved in stress-inducible geneexpression. As specified in the Background section, only one HSF hasbeen identified in yeast, Drosophila, and C. elegans, and three HSFshave been identified in vertebrates (Wu, Ann. Rev. Cell Dev. Biol. 1995;11:441-469; Morimoto, Genes and Development, 1998, 12: 3788-3796; Nakai,Cell Stress Chaperones, 1999, 4:86-86-93; Nover et al., Cell StressChaperones, 2001, 6: 177-189; recently, a fourth, HSF-like open readingframe (ORF) (HsfY) encoded on the Y chromosome was identified invertebrates—see Tessari et al., Mol. Human. Reprod., 2004, 10: 253-258).In human cells, three HSFs (HSF1, HSF2, and HSF4) have beencharacterized (Morimoto, et al., Genes Dev. 1998; 12:3788-3796). HSF1 isubiquitously expressed and plays the principle role in thestress-induced expression of HSPs. It is an apparent functional analogof Drosophila HSF. In contrast to few well-defined HSFs in other classesof organisms, plants are characterized by a great complexity of theplant HSF family which is thought to allow a highly flexible andefficient response to rapid changes in environmental conditions thataccompany the stationary lifestyle of plants (Nover et al., Cell StressChaperones, 2001, 6: 177-189; Kotak et al., Plant J., 2004, 39: 98-112).There is also a high degree of specialization in the response ofspecific plant HSFs to particular stress conditions (Rizhsky et al.,Plant Physiol., 2004, 134: 1683-1696). The model plant Arabidopsisthaliana contains 21 HSF genes, as well as several genes encodingHSF-like proteins (The Arabidopsis Genome Initiative 2000, Nature, 408:796-815), more than 16 HSF genes were found in tomato (Bharti et al.,Plant Cell, 2004, 16: 1521-1535; Nover et al., Cell Stress Chaperones,2001, 6: 177-189; Bharti and Nover, Heat stress-induced signalling; inPlant signal transduction: Frontiers in molecular biology, Scheel andWastemack eds., Oxford Univ. Press, 2002, pp. 74-115), and many otherHSF genes were identified in rice, maize and other species (Goff et al.,Science, 2002, 296: 92-100; Yu et al. 2002, Science, 2002, 296: 79-92;Kotak et al., Plant J., 2004, 39: 98-112). Plant HSF genes are assignedto three different classes (classes A, B and C) according to theirunique structural characteristics (Nover et al., Cell Stress Chaperones,2001, 6: 177-189). Class A HSF proteins comprise the largest group ofHSFs; they contain an activation domain at the C-terminus and arethought to be involved in transcriptional activation. Class B and classC HSFs lack a defined activation domain and function as repressorsand/or co-regulators of HSFA (reviewed in Miller and Mittler, Annals ofBotany, 2006, 98: 279-288).

Under normal conditions, mammalian HSF1 exists in the cell as aninactive monomer. Following exposure to elevated temperature, HSF1trimerizes and apparently relocates to the nucleus where it binds tospecific sites in HSP promoters upstream of the transcription initiationsite (Wu, Ann. Rev. Cell Dev. Biol. 1995; 11:441-469; Westwood, et al.,Mol. Cell. Biol. 1993; 13:3481-3486; Westwood, et al., Nature 1991;353:822-827). Similarly, plant HSF1A undergoes trimerization and DNAbinding upon stress (Mishra et al., Genes Dev, 2002, 16: 1555-1567).

In the context of the present invention, the term “augment” meansenhancing or extending the duration of a function, or both. In relationto a function of a Heat Shock RNA (HSR1) molecule, the term “augment”refers without limitation to the ability to enhance interaction with HSFor eEF1A or both, or enhance HSF binding to DNA, or enhance theHSF-mediated transcription, to extend the duration of the HSF-mediatedtranscriptional activation.

Within the meaning of the present invention, the term “inhibit” is usedto refer to any level of reduction in a function or amount of amolecule. For example, when used in relation to HSF activation, the term“inhibit” may mean without limitation reduction in the formation orfunction of HSF/HSR1/eEF1A ternary complex, reduction in HSF nucleartransport, reduction in HSF DNA binding, reduction in interaction withany of the elements of the transcriptional machinery, etc. When used inrelation to a function of a Heat Shock RNA (HSR1) molecule, the term“inhibit” may mean without limitation reduction in the conformationalchange in HSR1 in response to stress, reduction in interaction of HSR1with HSF and/or eEF1A, reduction in the formation or function ofHSF/HSR1/eEF1A ternary complex, reduction in HSR1 nuclear transport,etc.

The phrase “increasing sensitivity of a cancer cell to a treatment” isused herein to refer to any detectable decrease in propagation and/orsurvival of a cancer cell subjected to a given treatment.

The term “age-related disease” is used in the present invention toencompass all types of diseases associated with normal as well aspremature cellular and organism aging, including without limitationatherosclerosis and neurodegenerative diseases such as Alzheimer'sdisease and Parkinson's disease.

The terms “polynucleotide” or “nucleotide sequence” mean a series ofnucleotide bases (also called “nucleotides”) in DNA and RNA, and meanany chain of two or more nucleotides. A nucleotide sequence typicallycarries genetic information, including the information used by cellularmachinery to make proteins and enzymes. These terms include double orsingle stranded genomic and cDNA, RNA, any synthetic and geneticallymanipulated polynucleotide, and both sense and anti-sensepolynucleotide. This includes single- and double-stranded molecules,i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids.

The polynucleotides herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for example,producing an non-coding (untranslated) RNA or a protein by activatingthe cellular functions involved in transcription and translation of acorresponding gene or DNA sequence. A DNA sequence is expressed in or bya cell to form an “expression product” such as RNA or a protein. Theexpression product itself, e.g. the resulting RNA or protein, may alsobe said to be “expressed” by the cell.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence can be introduced into a hostcell, so as to transform the host and clone the vector or promoteexpression of the introduced sequence. Vectors include plasmids,cosmids, phages, viruses, etc. Vectors may further comprise selectablemarkers.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, used or manipulated in any way, for theproduction of a substance by the cell, for example, the expression bythe cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or other assays, as describedinfra.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 8, preferably no more than 100 nucleotides, thatis hybridizable to a genomic DNA molecule, a cDNA molecule, or an RNAmolecule. Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. In one embodiment, a labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. In another embodiment,oligonucleotides can be used as PCR primers, either for cloning or fordetection of a specific nucleic acid. In a further embodiment, anoligonucleotide of the invention can be used as antisenseoligonucleotides to inhibit a function or expression of a nucleic acidmolecule (see below). Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A sequence “encoding” an expression product, such as an RNA,polypeptide, protein, or enzyme, is a minimum nucleotide sequence that,when expressed, results in the production of that RNA, polypeptide,protein, or enzyme.

As used herein, the term “gene” means a DNA sequence that codes for aparticular non-coding (untranslated) RNA or a sequence of amino acids,which comprise all or part of one or more proteins or enzymes, and mayinclude regulatory (non-transcribed) DNA sequences, such as promotersequences, which determine for example the conditions under which thegene is expressed.

The term “antisense” nucleic acid molecule or oligonucleotide is used inthe present disclosure to refer to a single stranded (ss) nucleic acidmolecule, which may be DNA, RNA, a DNA-RNA chimera, or a derivativethereof, which, upon hybridizing under physiological conditions withcomplementary bases in an RNA or DNA molecule of interest, inhibits oractivates (in the case of “activating antisense oligonucleotides”) theexpression of the corresponding gene by modulating, e.g., RNAtranscription, RNA processing, RNA transport, mRNA translation, or RNAstability. As presently used, “antisense” broadly includes RNA-RNAinteractions, RNA-DNA interactions, and RNase-H mediated arrest.Antisense nucleic acid molecules can be encoded by a recombinant genefor expression in a cell (see, e.g., U.S. Pat. Nos. 5,814,500 and5,811,234), or alternatively they can be prepared synthetically (see,e.g., U.S. Pat. No. 5,780,607). According to the present invention, thefunction(s) of HSR1 in a cellular stress response may be identified,modulated and studied using antisense nucleic acids derived on the basisof HSR1 nucleic acid molecules of the invention. Furthermore, asdisclosed herein, due to their ability to modulate (inhibit or augment)HSR1 function(s), HSR1-specific antisense oligonucleotides may be usefulas therapeutics to treat cancer, inflammation, ischemia,neurodegeneration, age-related diseases, HIV infection, deafness, andrelated disorders or as tools for generating stress-resistant plants.

Specific examples of synthetic antisense oligonucleotides envisioned forthis invention include oligonucleotides that contain phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are those with CH₂—NH—O—CH₂,CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ andO—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—PO₂—O—CH₂). U.S.Pat. No. 5,677,437 describes heteroaromatic oligonucleoside linkages.Nitrogen linkers or groups containing nitrogen can also be used toprepare oligonucleotide mimics (U.S. Pat. Nos. 5,792,844 and 5,783,682).U.S. Pat. No. 5,637,684 describes phosphoramidate andphosphorothioamidate oligomeric compounds. Also envisioned areoligonucleotides having morpholino backbone structures (U.S. Pat. No.5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA)backbone, the phosphodiester backbone of the oligonucleotide may bereplaced with a polyamide backbone, the bases being bound directly orindirectly to the aza nitrogen atoms of the polyamide backbone (Nielsenet al., Science 1991; 254:1497). Other synthetic oligonucleotides maycontain substituted sugar moieties comprising one of the following atthe 2′ position: OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃where n is from 1 to about 10; C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-; S-, or N-alkyl;O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted sialyl; a fluorescein moiety; an RNA cleaving group; areporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Oligonucleotides may alsohave sugar mimetics such as cyclobutyls or other carbocyclics in placeof the pentofuranosyl group. Nucleotide units having nucleosides otherthan adenosine, cytidine, guanosine, thymidine and uridine may be used,such as inosine. In other embodiments, locked nucleic acids (LNA) can beused (reviewed in, e.g., Jepsen and Wengel, Curr. Opin. Drug Discov.Devel. 2004; 7:188-194; Crinelli et al., Curr. Drug Targets 2004;5:745-752). LNA are nucleic acid analog(s) with a 2′-O, 4′-C methylenebridge. This bridge restricts the flexibility of the ribofuranose ringand locks the structure into a rigid C3-endo conformation, conferringenhanced hybridization performance and exceptional biostability. LNAallows the use of very short oligonucleotides (less than 10 bp) forefficient hybridization in vivo.

The term “RNA interference” or “RNAi” refers to the ability of doublestranded RNA (dsRNA) to suppress the expression of a specific gene ofinterest in a homology-dependent manner. It is currently believed thatRNA interference acts post-transcriptionally by targeting RNA moleculesfor degradation. RNA interference commonly involves the use of dsRNAsthat are greater than 500 bp; however, it can also be mediated throughsmall interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs), whichcan be 10 or more nucleotides in length and are typically 18 or morenucleotides in length. For reviews, see Bosner and Labouesse, NatureCell Biol. 2000; 2:E31-E36 and Sharp and Zamore, Science 2000;287:2431-2433.

As used herein, the term “triplex-forming oligonucleotide” or “triplehelix forming oligonucleotide” or “TFO” refers to molecules that bind inthe major groove of duplex DNA and by so doing produce triplexstructures. TFOs bind to the purine-rich strand of the duplex throughHoogsteen or reverse Hoogsteen hydrogen bonding. They exist in twosequence motifs, either pyrimidine or purine. According to the presentinvention, TFOs can be employed as an alternative to antisenseoligonucleotides and can be both inhibitory and stimulatory. TFOs havealso been shown to produce mutagenic events, even in the absence oftethered mutagens. TFOs can increase rates of recombination betweenhomologous sequences in close proximity. TFOs of the present inventionmay be conjugated to active molecules. (for review see Casey and Glazer,Prog. Nucleic Acid. Res. Mol. Biol. 2001; 67:163-92)

The term “ribozyme” is used herein to refer to a catalytic RNA moleculecapable of mediating catalytic reactions on (e.g., cleaving) RNAsubstrates. Ribozyme specificity is dependent on complementary RNA-RNAinteractions (for a review, see Cech and Bass, Annu. Rev. Biochem. 1986;55:599-629). Two types of ribozymes, hammerhead and hairpin, have beendescribed. Each has a structurally distinct catalytic center. Thepresent invention contemplates the use of ribozymes designed on thebasis of the HSR1 or HSR1-encoding nucleic acid molecules of theinvention to induce catalytic reaction (e.g., cleavage) of the HSR1,thereby modulating (e.g., inhibiting) a function or expression of HSR1.Ribozyme technology is described further in Intracellular RibozymeApplications: Principals and Protocols, Rossi and Couture ed., HorizonScientific Press, 1999.

The term “nucleic acid hybridization” refers to anti-parallel hydrogenbonding between two single-stranded nucleic acids, in which A pairs withT (or U if an RNA nucleic acid) and C pairs with G. Nucleic acidmolecules are “hybridizable” to each other when at least one strand ofone nucleic acid molecule can form hydrogen bonds with the complementarybases of another nucleic acid molecule under defined stringencyconditions. Stringency of hybridization is determined, e.g., by (i) thetemperature at which hybridization and/or washing is performed, and (ii)the ionic strength and (iii) concentration of denaturants such asformamide of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two strands containsubstantially complementary sequences. Depending on the stringency ofhybridization, however, some degree of mismatches may be tolerated.Under “low stringency” conditions, a greater percentage of mismatchesare tolerable (i.e., will not prevent formation of an anti-parallelhybrid). See Molecular Biology of the Cell, Alberts et al., 3rd ed., NewYork and London: Garland Publ., 1994, Ch. 7.

Typically, hybridization of two strands at high stringency requires thatthe sequences exhibit a high degree of complementarity over an extendedportion of their length. Examples of high stringency conditions include:hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at65° C., followed by washing in 0.1×SSC/0.1% SDS (where 1×SSC is 0.15 MNaCl, 0.15 M Na citrate) at 68° C. or for oligonucleotide moleculeswashing in 6×SSC/0.5% sodium pyrophosphate at about 37° C. (for 14nucleotide-long oligos), at about 48° C. (for about 17 nucleotide-longoligos), at about 55° C. (for 20 nucleotide-long oligos), and at about60° C. (for 23 nucleotide-long oligos)).

Conditions of intermediate or moderate stringency (such as, for example,an aqueous solution of 2×SSC at 65° C.; alternatively, for example,hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at65° C. followed by washing in 0.2×SSC/0.1% SDS at 42° C.) and lowstringency (such as, for example, an aqueous solution of 2×SSC at 55°C.), require correspondingly less overall complementarity forhybridization to occur between two sequences. Specific temperature andsalt conditions for any given stringency hybridization reaction dependon the concentration of the target DNA and length and base compositionof the probe, and are normally determined empirically in preliminaryexperiments, which are routine (see Southern, J. Mol. Biol. 1975;98:503; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., vol. 2, ch. 9.50, CSH Laboratory Press, 1989; Ausubel et al.(eds.), 1989, Current Protocols in Molecular Biology, Vol. I, GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., New York, atp. 2.10.3).

As used herein, the term “standard hybridization conditions” refers tohybridization conditions that allow hybridization of two nucleotidemolecules having at least 50% sequence identity. According to a specificembodiment, hybridization conditions of higher stringency may be used toallow hybridization of only sequences having at least 75% sequenceidentity, at least 80% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, or at least 99% sequenceidentity.

Nucleic acid molecules that “hybridize” to any of the HSR1-encodingnucleic acids of the present invention may be of any length. In oneembodiment, such nucleic acid molecules are at least 10, at least 15, atleast 20, at least 30, at least 40, at least 50, and at least 70nucleotides in length. In another embodiment, nucleic acid moleculesthat hybridize are of about the same length as the particular HSR1 orHSR1-encoding nucleic acid.

The term “homologous” as used in the art commonly refers to therelationship between nucleic acid molecules or proteins that possess a“common evolutionary origin,” including nucleic acid molecules orproteins within superfamilies (e.g., the immunoglobulin superfamily) andnucleic acid molecules or proteins from different species (Reeck et al.,Cell 1987; 50:667). Such nucleic acid molecules or proteins havesequence homology, as reflected by their sequence similarity, whether interms of substantial percent similarity or the presence of specificresidues or motifs at conserved positions.

The terms “percent (%) sequence similarity”, “percent (%) sequenceidentity”, and the like, generally refer to the degree of identity orcorrespondence between different nucleotide sequences of nucleic acidmolecules or amino acid sequences of proteins that may or may not sharea common evolutionary origin (see Reeck et al., supra). Sequenceidentity can be determined using any of a number of publicly availablesequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.), etc.

To determine the percent identity between two amino acid sequences ortwo nucleic acid molecules, the sequences are aligned for optimalcomparison purposes. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences.In one embodiment, the two sequences are, or are about, of the samelength. The percent identity between two sequences can be determinedusing techniques similar to those described below, with or withoutallowing gaps. In calculating percent sequence identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990;87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA1993; 90:5873-5877. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al., J. Mol. Biol. 1990; 215:403.BLAST nucleotide searches can be performed with the NBLAST program,score=100, word length=12, to obtain nucleotide sequences homologous tosequences of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, word length=3, to obtain amino acidsequences homologous to protein sequences of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., Nucleic Acids Res. 1997; 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationship between molecules. See Altschul et al.(1997), supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on theWorldWideWeb. Another non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS 1988; 4:11-17. Such an algorithm is incorporated into theALIGN program (version 2.0), which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used.

In addition to the hamster HSR1 (SEQ ID NO: 1), human HSR1 (SEQ ID NO:37), Arabidopsis HSR1 (SEQ ID NO: 34), Drosophila HSR1 (SEQ ID NO: 33),and C. elegans HSR1 (SEQ ID NO: 36), the present invention furtherprovides polynucleotide molecules comprising nucleotide sequences havingcertain percentage sequence identities to these sequences. Suchsequences preferably hybridize under conditions of moderate or highstringency as described above, and may include species orthologs.

As used herein, the term “orthologs” refers to genes in differentspecies that apparently evolved from a common ancestral gene byspeciation. Normally, orthologs retain the same function through thecourse of evolution. Identification of orthologs can provide reliableprediction of gene function in newly sequenced genomes. Sequencecomparison algorithms that can be used to identify orthologs includewithout limitation BLAST, FASTA, DNA Strider, and the GCG pileupprogram. Orthologs often have high sequence similarity.

The present invention encompasses all orthologs of HSR1. Particularlyuseful HSR1 orthologs of the present invention are human, hamster,mouse, Xenopus, Drosophila, C. elegans, and yeast orthologs as well asvarious plant orthologs including, without limitation, Arabidopsis andcommercially important plants such as sugar storing and/orstarch-storing plants, for instance cereal species (rye, barley, oat,wheat, maize, millet, sago etc.), rice, pea, marrow pea, cassava, sugarcane, sugar beet, potato, tomato, cow pea or arrowroot, fiber-formingplants (e.g. flax, hemp, cotton), oil-storing plants (e.g. rape,sunflower), protein-storing plants (e.g. legumes, cereals, soybeans),vegetable plants (e.g. lettuce, chicory, Brassicaceae species such ascabbage), fruit trees, palms and other trees or wooden plants being ofeconomical value such as in forestry, forage plants (e.g. forage andpasture grasses, such as alfalfa, clover, ryegrass), and ornamentalplants (e.g. roses, tulips, hyacinths, camellias or shrubs).

The term “modulator” refers to a compound that differentially affectsthe expression or activity of a gene or gene product (e.g., RNA moleculeor protein), for example, in response to a stimulus (e.g., stress) thatnormally activates or represses the expression or activity of that geneor gene product when compared to the expression or activity of the geneor gene product not contacted with the stimulus. In one embodiment, thegene or gene product the expression or activity of which is beingmodulated includes a eukaryotic HSR1 or a gene or cDNA molecule thatencodes a eukaryotic HSR1. Examples of modulators of the HSR1 orHSR1-encoding nucleic acids of the present invention include withoutlimitation antisense nucleic acids, ribozymes, triple helix formingoligonucleotides (TFOs), and RNAi oligonucleotides.

A “test compound” is a molecule that can be tested for its ability toact as a modulator of a gene or gene product. Test compounds can beselected without limitation from small inorganic and organic molecules(i.e., those molecules of less than about 2 kDa, and more preferablyless than about 1 kDa in molecular weight), polypeptides (includingnative ligands, antibodies, antibody fragments, and other immunospecificmolecules), oligonucleotides, polynucleotide molecules, and derivativesthereof. In various embodiments of the present invention, a testcompound is tested for its ability to modulate a function of HSR1 ormodulate expression of HSR1. A compound that modulates a nucleic acid orprotein of interest is designated herein as a “candidate compound” or“lead compound” suitable for further testing and development. Candidatecompounds include, but are not necessarily limited to, the functionalcategories of agonist and antagonist.

An “agonist” is defined herein as a compound that interacts with (e.g.,binds to) a nucleic acid molecule or protein, and promotes, enhances,stimulates or potentiates the expression or function of the nucleic acidmolecule or protein. The term “partial agonist” is used to refer to anagonist which interacts with a nucleic acid molecule or protein, butpromotes only partial function of the nucleic acid molecule or protein.A partial agonist may also inhibit certain functions of the nucleic acidmolecule or protein with which it interacts. An “antagonist” interactswith (e.g., binds to) and inhibits or reduces the biological expressionor function of the nucleic acid molecule or protein.

The term “detectable change” as used herein in relation to a function orexpression level of a gene or gene product (e.g., HSR1) means anystatistically significant change and preferably at least a 1.5-foldchange as measured by any available technique such as electrophoreticgel mobility shift assays (EMSA), denaturing (8M urea) PAGE analysis,SDS-PAGE, Western blotting, nucleic acid hybridization, quantitativePCR, etc.

As used herein, the term “isolated” means that the material beingreferred to has been removed from the environment in which it isnaturally found, and is characterized to a sufficient degree toestablish that it is present in a particular sample. Suchcharacterization can be achieved by any standard technique, such as,e.g., sequencing, hybridization, immunoassay, functional assay,expression, size determination, or the like. Thus, a biological materialcan be “isolated” if it is free of cellular components, i.e., componentsof the cells in which the material is found or produced in nature. Anucleic acid molecule excised from the chromosome that it is naturally apart of is considered to be isolated. Such a nucleic acid molecule mayor may not remain joined to regulatory, or non-regulatory, or non-codingregions, or to other regions located upstream or downstream of the genewhen found in the chromosome. Nucleic acid molecules that have beenspliced into vectors such as plasmids, cosmids, artificial chromosomes,phages and the like are considered isolated. In a particular embodiment,a HSR1-encoding nucleic acid spliced into a recombinant vector, and/ortransformed into a host cell, is considered to be “isolated”.

Isolated nucleic acid molecules and isolated polynucleotide molecules ofthe present invention do not encompass uncharacterized clones inman-made genomic or cDNA libraries.

A protein that is associated with other proteins and/or nucleic acidswith which it is associated in an intact cell, or with cellularmembranes if it is a membrane-associated protein, is considered isolatedif it has otherwise been removed from the environment in which it isnaturally found and is characterized to a sufficient degree to establishthat it is present in a particular sample. A protein expressed from arecombinant vector in a host cell, particularly in a cell in which theprotein is not naturally expressed, is also regarded as isolated.

An isolated organelle, cell, or tissue is one that has been removed fromthe anatomical site (cell, tissue or organism) in which it is found inthe source organism.

An isolated material may or may not be “purified”. The term “purified”as used herein refers to a material (e.g., a nucleic acid molecule or aprotein) that has been isolated under conditions that detectably reduceor eliminate the presence of other contaminating materials. Contaminantsmay or may not include native materials from which the purified materialhas been obtained. A purified material preferably contains less thanabout 90%, less than about 75%, less than about 50%, less than about25%, less than about 10%, less than about 5%, or less than about 2% byweight of other components with which it was originally associated.

Methods for purification are well-known in the art. For example, nucleicacids or polynucleotide molecules can be purified by precipitation,chromatography (e.g., by affinity chromatography, preparative solidphase chromatography, oligonucleotide hybridization, and triple helixchromatography), ultracentrifugation, and other means. Polypeptides canbe purified by various methods including, without limitation,preparative disc-gel electrophoresis, isoelectric focusing, HPLC,reverse-phase HPLC, gel filtration, affinity chromatography, ionexchange and partition chromatography, precipitation and salting-outchromatography, extraction, and counter-current distribution. Cells canbe purified by various techniques, including centrifugation, matrixseparation (e.g., nylon wool separation), panning and otherimmunoselection techniques, depletion (e.g., complement depletion ofcontaminating cells), and cell sorting (e.g., fluorescence activatedcell sorting (FACS)). Other purification methods are possible. The term“substantially pure” indicates the highest degree of purity that can beachieved using conventional purification techniques currently known inthe art. In the context of analytical testing of the material,“substantially free” means that contaminants, if present, are below thelimits of detection using current techniques, or are detected at levelsthat are low enough to be acceptable for use in the relevant art, forexample, no more than about 2-5% (w/w). Accordingly, with respect to thepurified material, the term “substantially pure” or “substantially free”means that the purified material being referred to is present in acomposition where it represents 95% (w/w) or more of the weight of thatcomposition. Purity can be evaluated by chromatography, gelelectrophoresis, immunoassay, composition analysis, biological assay, orany other appropriate method known in the art.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within an acceptable standard deviation, per the practice inthe art. Alternatively, “about” can mean a range of up to ±20%,preferably up to ±10%, more preferably up to ±5%, and more preferablystill up to ±1% of a given value. Alternatively, particularly withrespect to biological systems or processes, the term can mean within anorder of magnitude, preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated, the term “about” is implicit and in this context meanswithin an acceptable error range for the particular value.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition. For example, in relation to cancer, the term “treat” maymean to relieve or alleviate at least one symptom selected from thegroup consisting of tumor growth, metastasis, sensitivity of tumor cellsto treatments such as chemotherapy, radiation therapy, thermotherapy,etc. Within the meaning of the present invention, the term “treat” alsodenotes to arrest, delay the onset (i.e., the period prior to clinicalmanifestation of a disease) and/or reduce the risk of developing orworsening a disease. The term “protect” is used herein to mean prevent,delay or treat, or all, as appropriate, development or continuance oraggravation of a disease in a subject. Within the meaning of the presentinvention, disease conditions include without limitation variouscancers, inflammation, ischemia/reperfusion injury, neurodegenerativedisorders, age-related diseases, deafness, and HIV infection.

As used herein the term “therapeutically effective” applied to dose oramount refers to that quantity of a compound or pharmaceuticalcomposition that is sufficient to result in a desired activity uponadministration to an animal in need thereof. Within the context of thepresent invention, the term “therapeutically effective” refers to thatquantity of a compound or pharmaceutical composition that is sufficientto reduce or eliminate at least one symptom of a cancer, inflammation,ischemia, neurodegenerative disorders, age-related diseases, HIVinfection, deafness, or related disorder. Note that when a combinationof active ingredients is administered the effective amount of thecombination may or may not include amounts of each ingredient that wouldhave been effective if administered individually.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to amammal (e.g., a human). Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in mammals, and moreparticularly in humans.

As used herein, the term “genetically modified” encompasses all animalsand plants into which an exogenous genetic material has been introducedand/or whose endogenous genetic material has been manipulated. Examplesof genetically modified animals and plants include without limitationtransgenic animals and plants, e.g., “knock-in” animals and plants withthe endogenous gene substituted with a heterologous gene or an orthologfrom another species or a mutated gene, “knockout” animals and plantswith the endogenous gene partially or completely inactivated, ortransgenic animals and plants expressing a mutated gene oroverexpressing a wild-type or mutated gene (e.g., upon targeted orrandom integration into the genome) and animals and plants containingcells harboring a non-integrated nucleic acid construct (e.g.,viral-based vector, antisense oligonucleotide, shRNA, siRNA, ribozyme,etc.), including animals and plants wherein the expression of anendogenous gene has been modulated (e.g., increased or decreased) due tothe presence of such construct.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. See, e.g., Sambrook, Fritsch andManiatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N. Y., 1989 (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984);Nucleic Acid Hybridization (Hames and Higgins eds. 1985); TranscriptionAnd Translation (Hames and Higgins eds. 1984); Animal Cell Culture(Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al.eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.1994; among others.

2. Novel Ternary Ribonucleoprotein Complex of the Invention

As specified in the Background Section, supra, heat shock transcriptionfactor HSF plays a central role in activation of the heat shock (HS)response in eukaryotic cells. Mammalian HSF1 (and its orthologs in otherorganisms) is present in unstressed cells in an inactive monomeric formand becomes activated by heat and other stress stimuli. Mammalian HSF1(and its orthologs in other organisms) activation involves trimerizationand acquisition of a site-specific DNA-binding activity.

As provided for the first time in the present invention, HSF activationby heat shock is mediated in vitro and in vivo by a ribonucleoproteinternary complex comprising translation elongation factor eEF1A and anovel RNA termed herein “Heat Shock RNA” or “HSR1”. eEF1A and HSR1mediate HSF activation upon stress under physiological conditions. Amongthe two HSF-associated factors, HSR1 serves as a cellular thermosensorthat determines the temperature threshold for the heat shock response.

Also provided herein are novel functional sequences of hamster HSR1cloned from BHK cells (SEQ ID NO: 1), human HSR1 cloned from HeLa cells(SEQ ID NO: 37), Arabidopsis HSR1 (SEQ ID NO: 34), Drosophila HSR1 (SEQID NO: 33), and C. elegans HSR1 (SEQ ID NO: 36, identical to DrosophilaHSR1) which all reveal very high degree of homology. HSR1 isconstitutively expressed in human, rodent, Drosophila, C. elegans andArabidopsis cells, and its homologs are functionally interchangeable.

HSR1 and eEF1A are both required for activation of HSF and constitute aminimal functional HSR1 activating complex. Indeed, as disclosed in theExamples Section, infra, in in vitro reconstituted system, RNAtranscribed in vitro from the cloned HSR1 can restore activation ofpurified HSF1 by the purified eEF1A. Neither eEF1A nor in vitrotranscribed HSR1 alone are capable of activating HSF1. However, whenboth components are added to HSF1 simultaneously, they induce HSF1binding to DNA. HSF1 activation by eEF1A and HSR1 is accompanied bytrimerization of the factor (see Examples, infra).

3. Novel Nucleic Acids of the Invention

The present invention provides an isolated ribonucleotide moleculecomprising a eukaryotic Heat Shock RNA (HSR1) sequence or a fragmentthereof. The invention also provides an isolated polynucleotide moleculeencoding a eukaryotic HSR1 or a fragment thereof. In one embodiment, theinvention provides isolated polynucleotide molecules comprising HSR1sequences from mammalian cells (e.g., human [e.g., HeLa], hamster [e.g.,BHK], mouse), Drosophila cells (e.g., Kc cells), C. elegans cells,Xenopus cells (e.g., Xenopus laevis), Arabidopsis cells (e.g.,Arabidopsis thaliana), and yeast (e.g., Saccharomyces cerevisiae) aswell as polynucleotide molecules comprising nucleotide sequencesencoding such HSR1s. In one embodiment, the present invention providesan isolated ribonucleotide molecule comprising hamster HSR1 cloned fromBHK cells having SEQ ID NO: 1. In another embodiment, the presentinvention provides an isolated ribonucleotide molecule comprising humanHSR1 cloned from HeLa cells having SEQ ID NO: 37. In a separateembodiment, the invention provides a 3′-truncated constitutively activefragment of human HSR1 (HSR1-435) having SEQ ID NO: 35. In yet anotherembodiment, the present invention provides an isolated ribonucleotidemolecule comprising HSR1 of Arabidopsis having SEQ ID NO: 34. In yetanother embodiment, the present invention provides an isolatedribonucleotide molecule comprising Drosophila HSR1 having SEQ ID NO: 33.In yet another embodiment, the present invention provides an isolatedribonucleotide molecule comprising C. elegans HSR1 having SEQ ID NO: 36,which is identical to Drosophila HSR1. As specified in Examples, infra,all of the above HSR1 sequences are highly homologous to each other. Infact, as specified in Example 6A, comparison of the Drosophila HSR1 (SEQID NO: 33) and human HSR1 (SEQ ID NO: 37) reveals only 8 nucleotidedifferences, at least some of which determine a temperature thresholdfor the heat shock response (i.e., 30-33° C. for Drosophila vs. 39-40°C. for humans). The in vitro results described in Example 6D and shownin FIG. 9C further demonstrate that the threshold temperature of HSF1activation is controlled by HSR1 making HSR1 a bona fide thermosensor.These results also demonstrate that Drosophila HSR1 and human HSR1 areinterchangeable in their ability to activate HSF1 at temperaturescorresponding to Drosophila and human heat shock thresholds,respectively.

In a separate embodiment, the invention also provides an isolatedpolynucleotide molecule (e.g., a gene or vector) encoding a eukaryoticHSR1 or a fragment thereof. Thus, in a specific embodiment, theinvention provides a human genomic sequence (SEQ ID NO: 32) comprisingthe sequence encoding human HSR1 and unique flanking sequences andflanking inverted repeats. In another embodiment, the invention providesan Arabidopsis genomic sequence (SEQ ID NO: 31) comprising the sequenceencoding Arabidopsis HSR1 with flanking inverted repeats.

The invention also provides an isolated single-stranded polynucleotidemolecule comprising a nucleotide sequence that is the complement of anucleotide sequence of one strand of any of the aforementionednucleotide sequences. In a specific embodiment, the present inventionprovides aHSR1 ribonucleotide molecule comprising a nucleotide sequence(SEQ ID NO: 3) that is the complement of hamster HSR1 having SEQ ID NO:1.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that is homologous to thenucleotide sequence of the HSR1 or the HSR1-encoding polynucleotidemolecule of the invention or fragments thereof (including, among others,constitutively active HSR1 fragments homologous to human HSR1-435). Theinvention also provides an isolated polynucleotide molecule comprising anucleotide sequence that hybridizes under standard hybridizationconditions to the polynucleotide molecule (or a complement thereof)encoding a eukaryotic HSR1 or a fragment thereof. As specified in theDefinitions Section, supra, examples of standard hybridizationconditions include without limitation (i) an aqueous solution of 2×SSC(where 1×SSC is 0.15 M NaCl, 0.15 M Na citrate) at 55° C. or 65° C. or(ii) hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mMEDTA at 65° C. followed by washing in 0.2×SSC/0.1% SDS at 42° C., etc.In a preferred embodiment, the homologous polynucleotide moleculehybridizes to the polynucleotide molecule comprising a nucleotidesequence of a eukaryotic HSR1 or a sequence that encodes a eukaryoticHSR1 or a fragment thereof under highly stringent conditions, such as,for example, (i) in an aqueous solution of 0.5×SSC at 65° C.; (ii)hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at65° C. followed by washing in 0.1×SSC/0.1% SDS at 68° C.; or foroligonucleotide molecules washing in 6×SSC/0.5% sodium pyrophosphate atabout 37° C. (for 14 nucleotide-long oligos), at about 48° C. (for about17 nucleotide-long oligos), at about 55° C. (for 20 nucleotide-longoligos), and at about 60° C. (for 23 nucleotide-long oligos)) (see theDefinitions Section, above). In a specific embodiment, the homologouspolynucleotide molecule hybridizes under highly stringent conditions tothe polynucleotide molecule (or a complement thereof) comprising anucleotide sequence SEQ ID NO: 1, SEQ ID NO: 37, SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 36, or a fragment thereof.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that is homologous to thenucleotide sequence of the HSR1 or the HSR1-encoding polynucleotidemolecule of the present invention. In a specific embodiment, suchpolynucleotide molecule has at least 50% sequence identity, preferablyat least 75% sequence identity, more preferably at least 90% sequenceidentity, and most preferably at least 95% sequence identity to at least100 consecutive nucleotides of the nucleotide sequence of the HSR1 orthe HSR1-encoding polynucleotide molecule of the present invention(e.g., as determined by a sequence comparison algorithm selected fromBLAST, FASTA, DNA Strider, and GCG, and preferably as determined by theBLAST program from the National Center for Biotechnology Information(NCBI-Version 2.2), available on the WorldWideWeb atncbi.nlm.nih.gov/BLAST/).

Also encompassed by the present invention are orthologs of thespecifically disclosed hamster, human, Drosophila, C. elegans, andArabidopsis HSR1 nucleic acids. Additional HSR1 orthologs can beidentified based on the sequences of hamster and human orthologsdisclosed herein, using standard sequence comparison algorithms such asBLAST, FASTA, DNA Strider, GCG, etc. Particularly useful HSR1 orthologsof the present invention are human, hamster, mouse, Xenopus, Drosophila,C. elegans, and yeast orthologs as well as various plant orthologsincluding, without limitation, Arabidopsis and commercially importantplants such as sugar storing and/or starch-storing plants, for instancecereal species (rye, barley, oat, wheat, maize, millet, sago etc.),rice, pea, marrow pea, cassaya, sugar cane, sugar beet, potato, tomato,cow pea or arrowroot, fiber-forming plants (e.g. flax, hemp, cotton),oil-storing plants (e.g. rape, sunflower), protein-storing plants (e.g.legumes, cereals, soybeans), vegetable plants (e.g. lettuce, chicory,Brassicaceae species such as cabbage), fruit trees, palms and othertrees or wooden plants being of economical value such as in forestry,forage plants (e.g. forage and pasture grasses, such as alfalfa, clover,ryegrass), and ornamental plants (e.g. roses, tulips, hyacinths,camellias or shrubs). In addition to sequence homology, the HSR1orthologs of the present invention possess at least one of the samefunctional properties of the hamster HSR1 or human HSR1 disclosed in theExamples Section, infra. Such properties include without limitation theability to interact with eEF1A, the ability to interact with HSF, theability to form a ternary complex with eEF1A and HSF, the ability toactivate HSF DNA binding, the ability to undergo a conformational changein response to stress, etc.

As disclosed above, the present invention provides polynucleotidemolecules consisting of nucleotide sequences that are fragments of thenucleotide sequence of any of the aforementioned HSR1-relatedpolynucleotide molecules of the present invention, or the complements ofsuch nucleotide sequences. Such fragments comprise at least about 5%, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, or at leastabout 99% of the contiguous nucleotide sequence of the HSR1-relatedpolynucleotide molecules of the present invention, or the complements ofsuch nucleotide sequences. Such fragments can be used for a variety ofpurposes including, e.g., to produce a portion of an HSR1 in anappropriate expression system to identify functional or structuraldomains of HSR1, to use it to inhibit HSR1 function (see the disclosureof antisense technology, infra), to prepare a hybridization probe, etc.In a separate embodiment, the present invention provides a 3′-truncatedconstitutively active fragment of human HSR1 (HSR1-435) having SEQ IDNO: 35 as well as other constitutively active fragments of human HSR1lacking 100-150 3′-terminal nucleotides and their constitutively activehomologs in other species.

In addition to the nucleotide sequences of any of the aforementionedHSR1-related polynucleotide molecules, polynucleotide molecules of thepresent invention can further comprise, or alternatively may consist of,nucleotide sequences selected from those sequences that naturally flankan HSR1-encoding nucleotide sequence in the chromosome, includingregulatory sequences.

The polynucleotide molecules encompassed by the present inventionexclude uncharacterized clones in man-made genomic or cDNA libraries.

4. Oligonucleotides of the Invention

The present invention further provides an oligonucleotide molecule thathybridizes to a polynucleotide molecule of the present invention, orthat hybridizes to a polynucleotide molecule having a nucleotidesequence that is the complement of a nucleotide sequence of apolynucleotide molecule of the present invention. Such anoligonucleotide molecule: (i) is about 10 nucleotides to about 200nucleotides in length, preferably from about 15 to about 100 nucleotidesin length, and more preferably about 20 to about 50 nucleotides inlength, and (ii) hybridizes to one or more of the polynucleotidemolecules of the present invention under highly stringent conditions(e.g., washing in 6×SSC/0.5% sodium pyrophosphate at about 37° C. forabout 14-base oligos, at about 48° C. for about 17-base oligos, at about55° C. for about 20-base oligos, and at about 60° C. for about 23-baseoligos). In one embodiment, an oligonucleotide molecule of the presentinvention is 100% complementary over its entire length to a portion ofat least one of the aforementioned polynucleotide molecules of thepresent invention. In another embodiment, an oligonucleotide molecule ofthe present invention is greater than 90% complementary over its entirelength to a portion of at least one of the aforementioned polynucleotidemolecules of the present invention.

Specific non-limiting examples of oligonucleotide molecules according tothe present invention include oligonucleotide molecules selected fromthe group consisting of SEQ ID NOS: 4-14 (see Examples Section, infra).

Oligonucleotide molecules of the present invention are useful for avariety of purposes, including, e.g., as hybridization probes fordetection or as PCR primers for amplification or detection ofHSR1-related polynucleotide molecules of the invention. Sucholigonucleotide molecules can be labeled, e.g., with radioactive labels(e.g., γ³²P), biotin, fluorescent labels, etc. In one embodiment, alabeled oligonucleotide molecule can be used as a probe to detect thepresence of a nucleic acid. For example, as disclosed in the ExamplesSection, infra, the invention provides a ribonucleotide (SEQ ID NO: 4)which hybridizes to region 167-405 of cloned hamster or human HSR1 andwhich can be used, e.g., as a Northern hybridization probe. In anotherembodiment, two oligonucleotide molecules (one or both of which may belabeled) can be used as PCR primers, either for cloning a full-lengthnucleic acid or a fragment of a nucleic acid encoding a gene product ofinterest, or to detect the presence of nucleic acids encoding a geneproduct. For example, as disclosed in the Examples Section, infra, theinvention provides HSR1-specific PCR primers (SEQ ID NOS: 5-6 and 17-19)that can be used for RT-PCR of hamster and human HSR1. Methods forconducting amplifications, such as the polymerase chain reaction (PCR),are described, among other places, in Saiki et al., Science 1988;239:487 and U.S. Pat. No. 4,683,202. Other amplification techniquesknown in the art, e.g., the ligase chain reaction, can alternatively beused (see, e.g., U.S. Pat. Nos. 6,335,184 and 6,027,923; Reyes et al.,Clinical Chemistry 2001; 47:131-40; and Wu et al., Genomics 1989;4:560-569).

The oligonucleotide molecules of the invention are also useful asantisense or short interfering (siRNA) or small hairpin (shRNA) RNAmolecules or triple helix forming oligonucleotides (TFOs) capable ofmodulating function or expression of HSR1 molecules of the invention(see below). Generally, oligonucleotide molecules are preparedsynthetically, preferably on a nucleic acid synthesizer, and may beprepared with non-naturally occurring phosphoester analog bonds, such asthioester bonds, where appropriate.

5. Recombinant Expression Systems a. Cloning and Expression Vectors

The present invention further provides compositions and constructs forcloning and expressing any of the polynucleotide molecules of thepresent invention, including cloning vectors, expression vectors,transformed host cells comprising any of said vectors, and novel strainsor cell lines derived therefrom. In one embodiment, the presentinvention provides a recombinant vector comprising a polynucleotidemolecule having a nucleotide sequence encoding a eukaryotic HSR1molecule. In a specific embodiment, the HSR1 molecule comprises theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 37, SEQ ID NO: 33, SEQID NO: 34, or SEQ ID NO: 36.

Recombinant vectors of the present invention, particularly expressionvectors, are preferably constructed so that the coding sequence for thepolynucleotide molecule of the present invention is in operativeassociation with one or more regulatory elements necessary fortranscription of the coding sequence to produce an HSR1. As used herein,the term “regulatory element” includes but is not limited to nucleotidesequences that encode inducible and non-inducible promoters, enhancers,operators and other elements known in the art that serve to drive and/orregulate expression of polynucleotide coding sequences.

Methods are known in the art for constructing recombinant vectorscontaining particular coding sequences in operative association withappropriate regulatory elements, and these can be used to practice thepresent invention. These methods include in vitro recombinanttechniques, synthetic techniques, and in vivo genetic recombination.See, e.g., the techniques described in Ausubel et al., 1989, above;Sambrook et al., 1989, above; Saiki et al., 1988, above; Reyes et al.,2001, above; Wu et al., 1989, above; U.S. Pat. Nos. 4,683,202; 6,335,184and 6,027,923.

A variety of expression vectors are known in the art that can beutilized to express a polynucleotide molecule of the present invention,including recombinant bacteriophage DNA, plasmid DNA, and cosmid DNAexpression vectors containing the particular coding sequences. Typicalprokaryotic expression vector plasmids that can be engineered to containa polynucleotide molecule of the present invention include pUC8, pUC9,pBR322 and pBR329 (Biorad Laboratories, Richmond, Calif.), pPL andpKK223 (Pharmacia, Piscataway, N. J.), pQE50 (Qiagen, Chatsworth,Calif.), and pGEM-T EASY (Promega, Madison, Wis.), pcDNA6.2/V5-DEST andpcDNA3.2/V5DEST (Invitrogen, Carlsbad, Calif.) among many others.Typical eukaryotic expression vectors that can be engineered to containa polynucleotide molecule of the present invention include anecdysone-inducible mammalian expression system (Invitrogen, Carlsbad,Calif.), cytomegalovirus promoter-enhancer-based systems (Promega,Madison, Wis.; Stratagene, La Jolla, Calif.; Invitrogen), andbaculovirus-based expression systems (Promega), among many others.

The regulatory elements of these and other vectors can vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription elements can beused. For instance, when cloning in mammalian cell systems, promotersisolated from the genome of mammalian cells, e.g., mouse metallothioneinpromoter, or from viruses that grow in these cells, e.g., vaccinia virus7.5 K promoter or Maloney murine sarcoma virus long terminal repeat, canbe used. Promoters obtained by recombinant DNA or synthetic techniquescan also be used to provide for transcription of the inserted sequence.In addition, expression from certain promoters can be elevated in thepresence of particular inducers, e.g., zinc and cadmium ions formetallothionein promoters. Non-limiting examples of transcriptionalregulatory regions or promoters include for bacteria, the β-galpromoter, the T7 promoter, the TAC promoter, trp and lac promoters,trp-lac fusion promoters, etc.; for yeast, glycolytic enzyme promoters,such as ADH-I and -II promoters, GPK promoter, PGI promoter, TRPpromoter, etc.; and for mammalian cells, SV40 early and late promoters,and adenovirus major late promoters, among others.

In the case of plant cells, expression control sequences may comprisepoly-A signals ensuring termination of transcription and stabilizationof the transcript, for example, those of the ³⁵S RNA from CauliflowerMosaic Virus (CaMV) or the nopaline synthase gene from Agrobacteriumtumefaciens. Additional regulatory elements may include transcriptionalas well as translational enhancers. A plant translational enhancer oftenused is the CaMV omega sequences. Similarly, the inclusion of an intron(e.g., intron-1 from the shrunken gene of maize) has been shown toincrease expression levels by up to 100-fold (Mait, Transgenic Research6 (1997), 143-156; Ni, Plant Journal 7 (1995), 661-676). The promotermay be homologous or heterologous with regard to its origin and/or withregard to the gene to be expressed. Suitable promoters are for instancethe promoter of the ³⁵S RNA of the CaMV (U.S. Pat. No. 5,352,605) andthe ubiquitin-promoter (U.S. Pat. No. 5,614,399) which lend themselvesto constitutive expression, the patatin gene promoter B33 (Rocha-Sosa,EMBO J. 8 (1989), 23-29) which lends itself to a tuber-specificexpression in potatoes or a promoter ensuring expression inphotosynthetically active tissues only, for instance the ST-LS1 promoter(Stockhaus, Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus,EMBO, J. 8 (1989) 2445-2451), the Ca/b-promoter (see for instance U.S.Pat. No. 5,656,496, U.S. Pat. No. 5,639,952, Bansal, Proc. Natl. Acad.Sci. USA 89 (1992), 3654-3658) and the Rubisco SSU promoter (U.S. Pat.Nos. 5,034,322 and 4,962,028) or the glutelin promoter from wheat whichlends itself to endosperm-specific expression (HOW promoter) (Anderson,Theoretical and Applied Genetics 96, (1998), 568-576; Thomas, Plant Cell2 (12), (1990), 1171-1180), the glutelin promoter from rice (Takaiwa,Plant Mol. Biol. 30(6) (1996), 1207-1221; Yoshihara, FESS Lett. 383(1996), 213-218; Yoshihara, Plant and Cell Physiology 37 (1996),107-111), the shrunken promoter from maize (Maas, EMBO J. 8 (11) (1990),3447-3452; Werr, Mol. Gen. Genet. 202(3) (1986), 471-475; Werr, Mol.Gen. Genet. 212(2), (1988), 342-350), the USP promoter, the phaseolinpromoter (Senqupta-Gopalan, Proc. Natl. Acad. Sci. USA 82 (1985),3320-3324; Bustos, Plant Cell 1 (9) (1989), 839-853) or promoters ofzein genes from maize (Pedersen, Cell 29 (1982), 1015-1026; Quatroccio,Plant Mol. Biol. 15 (1990), 81-93). However, promoters which are onlyactivated at a point in time determined by external influences can alsobe used (WO 93/07279; WO 00/29592; Lockinnon et al., Gene 33 (1985),137-149). Moreover, seed-specific promoters such as the USP promoterfrom Vicia faba which ensures a seed-specific expression (Fiedler, PlantMol. Biol. 22 (1993), 669-679; Baumlein, Mol. Gen. Genet. 225 (1991),459-467) or fruit-specific promoters such as described in WO 91/01373may be used. The polynucleotide to be expressed in plants may be alsolinked to a specific termination sequence which serves to terminatetranscription correctly and to add a poly-A-tail to the transcript (see,e.g., Gielen, EMBO J. 8 (1989) 23-29). Furthermore, if needed,polypeptide expression can in principle be targeted to anysub-localization of plant cells (e.g. cytosol, plastics, vacuole,mitochondria) or the plant (e.g. apoplast). In order to achieve thelocalization in a particular compartment, the coding region to beexpressed may be linked to DNA sequences encoding a signal sequence(also called “transit peptide”) ensuring localization in the respectivecompartment such as, for example, transit peptide of the plastidicFerredoxin: NADP oxidoreductase (FNR) of spinach (Jansen, CurrentGenetics 13 (1988), 517-522), transit peptide of the waxy protein ofmaize (Klosnen, Mol. Gen. Genet. 217 (1989), 155-161), signal peptidesof the ribulose bisphosphate carboxylase small subunit (Volter, Proc.Natl. Acad. Sci. USA 85 (1988), 846-850; Nawrath, Proc. Natl. Acad. Sci.USA 91 (1994), 12760-12764), signal peptides of the NADP malatdehydrogenase (Gallardo, Planta 197 (1995), 324-332), signal peptides ofthe glutathione reductase (Creissen, Plant J. 8 (1995), i67-175), signalpeptides of the R1 protein (Lorberth Nature Biotechnology 16, (1998),473-477), the vacuole-targeting N-terminal sequence of the patatinprotein (Sonnewald, Plant J. 1 (1991), 95-106), apoplast-targetingsignal sequence of the proteinase inhibitor 11-gene (Keil, Nucleic AcidRes. 14 (1986), 5641-5650; von Schaewen, EMBO J. 9 (1990), 30-33),apoplast-targeting signal sequence of the levansucrase gene from Erwiniaamylovora (Geier and Geider, Phys. Mol. Plant. Pathol. 42 (1993),387-404), apoplast-targeting signal sequence of a fragment of thepatatin gene B33 from Solanum tuberosum (Rosahl, Mol. Gen. Genet. 203(1986), 214-220); (see also targeting sequences described by Matsuokaand Neuhaus, Journal of Experimental Botany 50 (1999), 165-174;Chrispeels and Raikhel, Cell 68 (1992), 613-616; Matsuoka and Nakamura,Proc. Natl. Acad. Sci. USA 88 (1991), 834-838; Bednarek and Raikhel,Plant Cell 3 (1991), 1195-1206; Nakamura and Matsuoka (Plant Phys. 101(1993), 1-5; Braun, EMBO J. 11, (1992), 3219-3227; Oshima, Nucleic AcidRes. 18 (1990), 181).

Expression vectors can also be constructed that will express afusion/chimeric RNA comprising the HSR1 molecule of the presentinvention or a fragment thereof. Such fusion/chimeric can be used, e.g.,to study the functional and/or structural properties of the HSR1, or toaid in the identification or purification, or to improve the stability,of a recombinantly-expressed HSR1.

Expression vectors of the present invention also include variousexpression vectors for expression of HSF and eEF1A proteins (as well asany other molecules interacting with HSR1 and/or HSF/HSR1/eEF1A ternarycomplex). Possible fusion protein expression vectors include but are notlimited to vectors incorporating sequences that encode β-galactosidaseand trpE fusions, maltose-binding protein (MBP) fusions,glutathione-S-transferase (GST) fusions, polyhistidine fusions (e.g.,His₆ (SEQ ID NO: 56)), V5, HA, and myc. Methods known in the art can beused to construct expression vectors encoding these and other fusionproteins. In a specific embodiment, the present invention providesexpression vectors for production of fusion HSF or eEF1A proteins, e.g.,to assist their purification or detection. In non-limiting embodiments,e.g., an HSF- or eEF1A-MBP fusion protein can be purified using amyloseresin; an HSF- or eEF1A-GST fusion protein can be purified usingglutathione-agarose beads; and an HSF- or eEF1A-His₆ (SEQ ID NO: 56)fusion protein can be purified using divalent nickel resin.Alternatively, antibodies against a carrier protein or peptide can beused for affinity chromatography purification of the fusion protein. Forexample, a nucleotide sequence coding for the target epitope of amonoclonal antibody can be engineered into the expression vector inoperative association with the regulatory elements and situated so thatthe expressed epitope is fused to an HSF or eEF1A protein of the presentinvention. In a non-limiting embodiment, a nucleotide sequence codingfor the FLAG™ epitope tag (International Biotechnologies Inc.), which isa hydrophilic marker peptide, can be inserted by standard techniquesinto the expression vector at a point corresponding, e.g., to the aminoor carboxyl terminus of the HSF or eEF1A protein. The expressed HSF oreEF1A protein-FLAG™ epitope fusion product can then be detected andaffinity-purified using commercially available anti-FLAG™ antibodies.The expression vector can also be engineered to contain polylinkersequences that encode specific protease cleavage sites so that theexpressed HSF or eEF1A protein can be released from a carrier region orfusion partner by treatment with a specific protease. For example, thefusion protein vector can include a nucleotide sequence encoding athrombin or factor Xa cleavage site, among others.

To aid in the selection of host cells transformed or transfected with arecombinant vector of the present invention, the vector can beengineered to further comprise a coding sequence for a reporter geneproduct or other selectable marker. Such a coding sequence is preferablyin operative association with the regulatory elements, as describedabove. Reporter genes that are useful in practicing the invention areknown in the art, and include those encoding chloramphenicolacetyltransferase (CAT), green fluorescent protein (GFP), fireflyluciferase, and human growth hormone (hGH), among others. Nucleotidesequences encoding selectable markers are known in the art, and includethose that encode gene products conferring resistance to antibiotics oranti-metabolites, or that supply an auxotrophic requirement. Examples ofsuch sequences include those that encode thymidine kinase activity, orresistance to methotrexate, ampicillin, kanamycin, chloramphenicol,zeocin, pyrimethamine, aminoglycosides, hygromycin, blasticidine, orneomycin, among others.

b. Transformation of Host Cells

The present invention further provides a transformed host cellcomprising a polynucleotide molecule or recombinant vector of thepresent invention, and a cell line derived therefrom. Such host cellsare useful for cloning and/or expressing polynucleotide molecules of thepresent invention. Such transformed host cells include but are notlimited to microorganisms, such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeasttransformed with a recombinant vector, as well as transfected plantcells or animal cells, such as insect cells (e.g., Drosophila Kc cells)or mammalian cells (e.g., mouse, rat, hamster [e.g., BHK-21 cells ATCCAccession No. CRL-1632], mouse, cow, monkey, or human cells [e.g., HeLacells ATCC Accession No. CCL-2]). In a specific embodiment, the presentinvention provides HeLa cell lines stably expressing GFP (HS) orHSR1-directed siRNA (SEQ ID NO: 7 and SEQ ID NO: 27 [siHSR1-160] or SEQID NO: 20 and SEQ ID NO: 28 [siHSR1-224]) or antisense HSR1 (aHSR1; SEQID NO: 3).

The recombinant vector of the invention is preferably transformed ortransfected into one or more host cells of a substantially homogeneousculture of cells. The vector is generally introduced into host cells inaccordance with known techniques, such as, e.g., by protoplasttransformation, calcium phosphate precipitation, calcium chloridetreatment, microinjection, electroporation, transfection by contact witha recombined virus, liposome-mediated transfection, DEAE-dextrantransfection, transduction, conjugation, or microprojectile bombardment,among others. Selection of transformants can be conducted by standardprocedures, such as by selecting for cells expressing a selectablemarker, e.g., antibiotic resistance, associated with the recombinantexpression vector.

Methods for the introduction of foreign genes into plants are also wellknown in the art. These include, for example, the transformation ofplant cells or tissues with T-DNA using Agrobacterium tumefaciens orAgrobacterium rhizogenes, the fusion of protoplasts, direct genetransfer (see, e.g., EP-A 164 575), injection, electroporation, vacuuminfiltration, biolistic methods like particle bombardment,pollen-mediated transformation, plant RNA virus-mediated transformation,liposome-mediated transformation, transformation using wounded orenzyme-degraded immature embryos, or wounded or enzyme-degradedembryogenic callus and other methods known in the art. The vectors usedin the method of the invention may contain further functional elements,for example “left border”- and “right border”-sequences of the T DNA ofAgrobacterium which allow stable integration into the plant genome.Furthermore, methods and vectors are known to the person skilled in theart which permit the generation of marker free transgenic plants, i.e.the selectable or scorable marker gene is lost at a certain stage ofplant development or plant breeding. This can be achieved by, forexample co-transformation (Lvznik, Plant Mol. Biol. 13 (1989), 151-161;Penn, Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems whichutilize enzymes capable of promoting homologous recombination in plants(see, e.g., WO 97/08331; Bavley, Plant Mol. Biol. 18 (1992), 353-361);Llovd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet.230 (1991), 170-176; Onouchi, Muck Acids Res. 19 (1991), 6373-6378).Methods for the preparation of appropriate vectors are described by,e.g., Sambrook and Russell (2001), Molecular Cloning: A LaboratoryManual, CSH Press, Cold Spring Harbor, N. Y., USA. Suitable strains ofAgrobacterium tumefaciens and vectors as well as transformation ofAgrobacteria and appropriate growth and selection media are well knownto those skilled in the art and are described in the prior art (GV3101(pMK9ORK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic.Acid Res. 12 (1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989),8467-8471; Koncz, Plant Mol. Biol. (1992), 963-976; Koncz, Specializedvectors for gene tagging and expression studies. In: Plant MolecularBiology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, TheNetherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120 516; Hoekema:The Binary Plant Vector System, Offsetdrukkerij Kanters B. V.,Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46;An, EMBO J. 4 (1985), 277-287). Methods for the transformation usingbiolistic methods are well known to the person skilled in the art; see,e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11(1993), 1553-1558 and Christou Trends in Plant Science 1 (1996),423-431. Microinjection can be performed as described in Potrvkus andSpangenbern (ads.), Gene Transfer To Plants. Springer Verlag, Berlin,N.Y. (1995). The transformation of most dicotyledonous plants ispossible with the methods described above. But also for thetransformation of monocotyledonous plants several successfultransformation techniques have been developed. These include the;transformation using biolistic methods as, e.g., described above as wellas protoplast transformation, electroporation of partially permeabilizedcells, introduction of DNA using glass fibers, etc. Also, thetransformation of monocotyledonous plants by means ofAgrobacterium-based vectors has been described (Chan, Plant Mol. Biol.22 (1993), 491-506; Hiei, Plant J. 6 (1994) 271-282; Denn, Science inChina 33 (1990), 28-34; Wilmink, Plant Cell Reports 11 (1992), 76-80;May, Bio/Technology 13 (1995), 486-492; Conner and Dormisse, Int. J.Plant Sci. 153 (1992), 550-555; Ritchie, Transgenic Res. 2 (1993),252-265). An alternative system for transforming monocotyledonous plantsis the transformation by the biolistic approach (Wan and Lemaux, PlantPhysiol. 104 (1994), 37-48; Vasii, Bio/Technology 11 (1993), 1553-I1558; Ritala, Plant Mol. Biol. 24 (1994) 317-325; Spencer, Theor. Appl.Genet. 79 (1990), 625-631). The transformation of maize in particularhas been repeatedly described in the literature (see for instance WO95/06128, EP 0 513 849, EP 0 465 875, EP 29 24 35; Fromm, Biotechnology8, (1990), 833-844; Gordon-Kamm, Plant Cell 2, (1990), 603-618; Koziel,Biotechnology 11 (1993), 194-200; Moroc, Theor. Appl. Genet. 80, (1990),721-726). The successful transformation of other types of cereals hasalso been described for instance of barley (Wan and Lemaux, supra;Ritala, supra, Krens, Nature 296 (1982), 72-74), wheat (Nehra, Plant J.5 (1994), 285-297) and rice. For transformation of pasture grass, seeTrinh, Plant Cell Reports 17 (1998), 345-355; Hoffmann, MolecularPlant-Microbe Interactions 10 307-315; Altpeter, Molecular Breeding 6(2000), 519-528; Larkin, Transgenic Research (1996), 325-335, Voise Y,Plant Cell Reports 13 (1994), 309-314 or Tabe, J. Anim. Sci. 73 (1995),2752-2759. For transformation of sugar cane, see Bower and Birch, PlantJournal 2(3) (1992), 409-416. For transformation of sugar beet, see WO91/13159. For transformation of vegetable plants as exemplified fortomato, see Chvi and I Phillips, Plant Cell Reports 6 (1987), 105-108;Davis, Plant Cell, Tissue & Organ! Culture 24, 1991 115-121; van Roekel,Plant Cell Reports 12 (1993), 644-647. For sweet tomato, see Newell,Plant Science 107 (1995), 215-227. For transformation of potato, wheat,barley, rape, soybean, rice, maize, see Excerpts from the text book“Gene Transfer to Plants”, Springer Verlag, Lab Manual, 1995.

The resulting transformed plant cell can then be used to regenerate atransformed plant in a manner known by a skilled person.

Once an expression vector is introduced into the host cell, the presenceof the polynucleotide molecule of the present invention, eitherintegrated into the host cell genome or maintained episomally, can beconfirmed by standard techniques, e.g., by DNA-DNA, DNA-RNA, orRNA-antisense RNA hybridization analysis, restriction enzyme analysis,PCR analysis including reverse transcriptase PCR (RT-PCR), detecting thepresence of a “marker” gene function, or by immunological or functionalassay to detect the expected protein product.

6. Use of the Nucleic Acid Molecules of the Invention to Modulate HSR1Function and Expression

As disclosed above, the HSR1-related nucleic acid molecules of theinvention or the nucleic acid molecules comprising sequences thathybridize to them under standard hybridization conditions (includingtheir homologs/orthologs, complementary sequences and variousoligonucleotide probes and primers derived from them) can be used tomodulate (e.g., inhibit or augment) a function of HSR1 or HSF/HSR1/eEF1Aternary complex (e.g., by modulating interaction between HSR1 and eEF1A,interaction between HSR1 and HSF, formation of a ternary complexHSF/HSR1/eEF1A, activation of HSF DNA binding, the ability of HSR1 toundergo a conformational change in response to stress, etc.). TheHSR1-related nucleic acid molecules of the invention or the nucleic acidmolecules comprising sequences that hybridize to them under standardhybridization conditions (including their homologs/orthologs,complementary sequences and various oligonucleotide probes and primersderived from them) can be also used to modulate expression of HSR1 genes(e.g., by augmenting or inhibiting transcription, processing, transport,or by blocking or promoting degradation of corresponding RNAs).

In a specific embodiment (see more details below), the present inventionprovides HSR1-specific antisense oligonucleotides, RNA interference(RNAi) molecules, ribozymes, and triple helix forming oligonucleotides(TFOs) which can be effectively used to mediate any of these functions.In conjunction with these antisense oligonucleotides, RNA interference(RNAi) molecules, ribozymes, and triple helix forming oligonucleotides(TFOs), the present invention provides a method of modulating (e.g.,inhibiting or increasing) a stress tolerance in a cell comprisingadministering said molecules to the cell.

Also, as specified in greater detail below, the antisenseoligonucleotides, RNA interference (RNAi) molecules, ribozymes, andtriple helix forming oligonucleotides (TFOs) of the invention can beused as a basis for developing stress-resistant plants and therapeuticsto treat cancer, inflammation, ischemia, neurodegenerative disorders,age-related diseases, HIV infection, deafness, and related disorders.

a. Antisense Nucleic Acids

As specified in Section 4, above, to achieve modulation of a function ofan HSR1 and/or HSF/HSR1/eEF1A ternary complex or modulation ofexpression of an HSR1 gene, the nucleic acid molecules of the inventioncan be used to design antisense oligonucleotides. Antisenseoligonucleotides of the invention comprise from about 6 to about 200nucleotides, but are typically 13 to 50 nucleotides in length. Forexample, Examples Section, infra, provides four specific 45-merantisense oligonucleotides corresponding to the cloned hamster HSR1 (SEQID NO: 1), i.e., 1^(HSR1) (SEQ ID NO: 9; complementary to HSR1 [SEQ IDNO: 1] nt 1-44), 2^(HSR1) (SEQ ID NO: 10; complementary to HSR1 [SEQ IDNO: 1] nt 40-84), 5^(HSR1) (SEQ ID NO: 11; complementary to HSR1 [SEQ IDNO: 1] nt 157-201), 6^(HSR1) (SEQ ID NO: 12; complementary to HSR1 [SEQID NO: 1] nt 196-240), which produce significant inhibition of HSFactivation in the in vitro reconstituted system. Furthermore, asdisclosed in the Examples, when transfected in cultured cells (e.g., BHKand HeLa), phosphothioate derivative of the oligonucleotide 6^(HSR1)impairs the HS response and suppresses the induction of thermotolerancein vivo by producing a significant decrease in the level of HSF DNAbinding activity and blocking the synthesis of HSP72 protein after heatshock. In addition to the antisense oligonucleotides which inhibit HSFactivation and impair HS response, the present invention alsoencompasses antisense oligonucleotides which stimulate HSF activationand HS response. Such “activating” antisense oligonucleotides mayfunction by changing HSR1 conformation into a conformation available forHSF activation.

The antisense oligonucleotides of the invention comprise sequencescomplementary to at least a portion of the corresponding HSR1 orHSR1-encoding nucleic acid. However, 100% sequence complementarity isnot required so long as formation of a stable duplex (for singlestranded antisense oligonucleotides) or triplex (for double strandedantisense oligonucleotides) can be achieved. The ability to hybridizewill depend on both the degree of complementarity and the length of theantisense oligonucleotides. Generally, the longer the antisenseoligonucleotide, the more base mismatches with the corresponding nucleicacid target can be tolerated. One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

The antisense oligonucleotides can be DNA or RNA or chimeric mixtures,or derivatives or modified versions thereof, and can be single-strandedor double-stranded. The antisense oligonucleotides can be modified atthe base moiety, sugar moiety, or phosphate backbone, or a combinationthereof. For example, a HSR1-specific antisense oligonucleotide cancomprise at least one modified base moiety selected from a groupincluding but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

In another embodiment, the HSR1-specific antisense oligonucleotidecomprises at least one modified sugar moiety, e.g., a sugar moietyselected from arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the HSR1-specific antisense oligonucleotidecomprises at least one modified phosphate backbone selected from aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof. In a specificembodiment, the present invention provides phosphorothioate antisenseoligonucleotides (e.g., 6^(HSR1) and anti-6^(HSR1) phosphothioatemodified at 3′ and 5′ ends to increase their stability) and chimerasbetween methylphosphonate and phosphodiester oligonucleotides. Theseoligonucleotides appear to provide good in vivo activity due tosolubility, nuclease resistance, good cellular uptake, ability toactivate RNase H, and high sequence selectivity.

The antisense oligonucleotide can include other appending groups such aspeptides, or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 1989;86:6553-6556; Lemaitre et al., Proc. Natl. Acad. Sci. USA 1987;84:648-652; PCT Publication No. WO 88/09810) or blood-brain barrier(see, e.g., PCT Publication No. WO 89/10134), hybridization-triggeredcleavage agents (see, e.g., Krol et al., BioTechniques 1988; 6:958-976),intercalating agents (see, e.g., Zon, Pharm. Res. 1988; 5:539-549), etc.

In another embodiment, the antisense oligonucleotide can includeα-anomeric oligonucleotides. An α-anomeric oligonucleotide formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 1987; 15:6625-6641).

In yet another embodiment, the antisense oligonucleotide can be amorpholino antisense oligonucleotide (i.e., an oligonucleotide in whichthe bases are linked to 6-membered morpholine rings, which are connectedto other morpholine-linked bases via non-ionic phosphorodiamidateintersubunit linkages). Morpholino oligonucleotides are highly resistantto nucleases and have good targeting predictability, high in-cellefficacy and high sequence specificity (U.S. Pat. No. 5,034,506;Summerton, Biochim. Biophys. Acta 1999; 1489:141-158; Summerton andWeller, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195; Arora et al.,J. Pharmacol. Exp. Ther. 2000; 292:921-928; Qin et al., AntisenseNucleic Acid Drug Dev. 2000; 10:11-16; Heasman et al., Dev. Biol. 2000;222:124-134; Nasevicius and Ekker, Nat. Genet. 2000; 26:216-220).

Antisense oligonucleotides of the invention may be chemicallysynthesized, for example using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer. Antisensenucleic acid oligonucleotides of the invention can also be producedintracellularly by transcription from an exogenous sequence. Forexample, a vector can be introduced in vivo such that it is taken up bya cell within which the vector or a portion thereof is transcribed toproduce an antisense RNA. Such a vector can remain episomal or becomechromosomally integrated, so long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. In another embodiment, “naked” antisensenucleic acids can be delivered to adherent cells via “scrape delivery”,whereby the antisense oligonucleotide is added to a culture of adherentcells in a culture vessel, the cells are scraped from the walls of theculture vessel, and the scraped cells are transferred to another platewhere they are allowed to re-adhere. Scraping the cells from the culturevessel walls serves to pull adhesion plaques from the cell membrane,generating small holes that allow the antisense oligonucleotides toenter the cytosol.

b. RNA Interference (RNAi)

RNA interference (RNAi) is a process of sequence-specificpost-transcriptional gene silencing by which double stranded RNA (dsRNA)homologous to a target locus can specifically inactivate gene functionin plants, fungi, invertebrates, and vertebrates, including mammals(Hammond et al., Nature Genet. 2001; 2:110-119; Sharp, Genes Dev. 1999;13:139-141). This dsRNA-induced gene silencing is mediated by shortdouble-stranded small interfering RNAs (siRNAs) generated from longerdsRNAs by ribonuclease III cleavage (Bernstein et al., Nature 2001;409:363-366 and Elbashir et al., Genes Dev. 2001; 15:188-200).RNAi-mediated gene silencing is thought to occur via sequence-specificRNA degradation, where sequence specificity is determined by theinteraction of an siRNA with its complementary sequence within a targetRNA (see, e.g., Tuschl, Chem. Biochem. 2001; 2:239-245).

For mammalian systems, RNAi commonly involves the use of dsRNAs that aregreater than 500 bp; however, it can also be activated by introductionof either siRNAs (Elbashir, et al., Nature 2001; 411: 494-498) or shorthairpin RNAs (shRNAs) bearing a fold back stem-loop structure (Paddisonet al., Genes Dev. 2002; 16: 948-958; Sui et al., Proc. Natl. Acad. Sci.USA 2002; 99:5515-5520; Brummelkamp et al., Science 2002; 296:550-553;Paul et al., Nature Biotechnol. 2002; 20:505-508).

The siRNAs to be used in the methods of the present invention arepreferably short double stranded nucleic acid duplexes comprisingannealed complementary single stranded nucleic acid molecules. Inpreferred embodiments, the siRNAs are short dsRNAs comprising annealedcomplementary single strand RNAs. However, the invention alsoencompasses embodiments in which the siRNAs comprise an annealed RNA:DNAduplex, wherein the sense strand of the duplex is a DNA molecule and theantisense strand of the duplex is a RNA molecule.

Preferably, each single stranded nucleic acid molecule of the siRNAduplex is of from about 19 nucleotides to about 27 nucleotides inlength. In preferred embodiments, duplexed siRNAs have a 2 or 3nucleotide 3′ overhang on each strand of the duplex. In preferredembodiments, siRNAs have 5′-phosphate and 3′-hydroxyl groups.

The RNAi molecules to be used in the methods of the present inventioncomprise nucleic acid sequences that are complementary to the nucleicacid sequence of a portion of the target HSR1 or HSR1-encoding nucleicacid. In certain embodiments, the portion of the target nucleic acid towhich the RNAi probe is complementary is at least about 15 nucleotidesin length. The target locus to which an RNAi probe is complementary mayrepresent a transcribed portion of the HSR1 gene or an untranscribedportion of the HSR1 gene (e.g., intergenic regions, repeat elements,etc.). As disclosed in the Examples Section, infra, the presentinvention provides HSR1-directed siRNAs, siHSR1-160 (SEQ ID NO: 7 andSEQ ID NO: 27) and siHSR1-224 (SEQ ID NO: 20 and SEQ ID NO: 28;corresponding to hamster HSR1 [SEQ ID NO: 1] nt 196-240), andcorresponding mutant siRNAs, mut160 (C11→G; SEQ ID NO: 8 and SEQ ID NO:29) and mut224 (G4→C; SEQ ID NO: 21 and SEQ ID NO: 30).

The RNAi molecules may include one or more modifications, either to thephosphate-sugar backbone or to the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one heteroatom other than oxygen, such as nitrogen or sulfur. Inthis case, for example, the phosphodiester linkage may be replaced by aphosphothioester linkage. Similarly, bases may be modified to block theactivity of adenosine deaminase. Where the RNAi molecule is producedsynthetically, or by in vitro transcription, a modified ribonucleosidemay be introduced during synthesis or transcription.

According to the present invention, siRNAs may be introduced to a targetcell as an annealed duplex siRNA, or as single stranded sense andantisense nucleic acid sequences that, once within the target cell,anneal to form the siRNA duplex. Alternatively, the sense and antisensestrands of the siRNA may be encoded on an expression construct that isintroduced to the target cell. Upon expression within the target cell,the transcribed sense and antisense strands may anneal to reconstitutethe siRNA.

The shRNAs to be used in the methods of the present invention comprise asingle stranded “loop” region connecting complementary inverted repeatsequences that anneal to form a double stranded “stem” region.Structural considerations for shRNA design are discussed, for example,in McManus et al., RNA 2002; 8:842-850. In certain embodiments the shRNAmay be a portion of a larger RNA molecule, e.g., as part of a larger RNAthat also contains U6 RNA sequences (Paul et al., supra).

In preferred embodiments, the loop of the shRNA is from about 1 to about9 nucleotides in length. In preferred embodiments the double strandedstem of the shRNA is from about 19 to about 33 base pairs in length. Inpreferred embodiments, the 3′ end of the shRNA stem has a 3′ overhang.In particularly preferred embodiments, the 3′ overhang of the shRNA stemis from 1 to about 4 nucleotides in length. In preferred embodiments,shRNAs have 5′-phosphate and 3′-hydroxyl groups.

Although the RNAi molecules useful according to the invention preferablycontain nucleotide sequences that are fully complementary to a portionof the target nucleic acid, 100% sequence complementarity between theRNAi probe and the target nucleic acid is not required to practice theinvention.

Similar to the above-described antisense oligonucleotides, RNAimolecules of the invention can be synthesized by standard methods knownin the art, e.g., by use of an automated synthesizer. RNAs produced bysuch methodologies tend to be highly pure and to anneal efficiently toform siRNA duplexes or shRNA hairpin stem-loop structures. Followingchemical synthesis, single stranded RNA molecules are deprotected,annealed to form siRNAs or shRNAs, and purified (e.g., by gelelectrophoresis or HPLC). Alternatively, standard procedures may usedfor in vitro transcription of RNA from DNA templates carrying RNApolymerase promoter sequences (e.g., T7 or SP6 RNA polymerase promotersequences). Efficient in vitro protocols for preparation of siRNAs usingT7 RNA polymerase have been described (Donzé and Picard, Nucleic AcidsRes. 2002; 30:e46; and Yu et al., Proc. Natl. Acad. Sci. USA 2002;99:6047-6052). Similarly, an efficient in vitro protocol for preparationof shRNAs using T7 RNA polymerase has been described (Yu et al., supra).The sense and antisense transcripts may be synthesized in twoindependent reactions and annealed later, or may be synthesizedsimultaneously in a single reaction.

RNAi molecules may be formed within a cell by transcription of RNA froman expression construct introduced into the cell. For example, both aprotocol and an expression construct for in vivo expression of siRNAsare described in Yu et al., supra. Similarly, protocols and expressionconstructs for in vivo expression of shRNAs have been described(Brummelkamp et al., supra; Sui et al., supra; Yu et al., supra; McManuset al., supra; Paul et al., supra).

The expression constructs for in vivo production of RNAi moleculescomprise RNAi encoding sequences operably linked to elements necessaryfor the proper transcription of the RNAi encoding sequence(s), includingpromoter elements and transcription termination signals. Preferredpromoters for use in such expression constructs include thepolymerase-III HI-RNA promoter (see, e.g., Brummelkamp et al., supra)and the U6 polymerase-III promoter (see, e.g., Sui et al., supra; Paul,et al. supra; and Yu et al., supra). The RNAi expression constructs canfurther comprise vector sequences that facilitate the cloning of theexpression constructs. Standard vectors that maybe used in practicingthe current invention are known in the art (e.g., pSilencer 2.0-U6vector, Ambion Inc., Austin, Tex.).

c. Ribozyme Modulation

In another embodiment, a function of HSR1 and/or HSF/HSR1/eEF1A ternarycomplex or expression of HSR1 genes of the present invention can bemodulated by ribozymes designed based on the nucleotide sequencethereof.

Ribozymes are enzymatic RNA molecules capable of catalyzing thesequence-specific cleavage of RNA (for a review, see Rossi, CurrentBiology 1994; 4:469-471). The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage event.The composition of ribozyme molecules must include: (i) one or moresequences complementary to the target RNA; and (ii) a catalytic sequenceresponsible for RNA cleavage (see, e.g., U.S. Pat. No. 5,093,246).

According to the present invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave RNAs at locations dictated byflanking regions that form complementary base pairs with the target RNA.The sole requirement is that the target RNA has the following sequenceof two bases: 5′-UG-3′. The construction of hammerhead ribozymes isknown in the art, and described more fully in Myers, Molecular Biologyand Biotechnology: A Comprehensive Desk Reference, VCH Publishers, NewYork, 1995 (see especially FIG. 4, page 833) and in Haseloff andGerlach, Nature 1988; 334:585-591.

Preferably, the ribozymes of the present invention which act to inhibitHSR1 function are engineered so that the cleavage recognition site islocated near the 5′ end of HSR1, i.e., to increase efficiency andminimize the intracellular accumulation of non-functional HSR1molecules.

The present invention encompasses both inhibitory and “activating”ribozyme molecules. Activating ribozyme molecules can be designed, e.g.,to eliminate a potentially inhibitory domain(s) of HSR1.

As in the case of antisense oligonucleotides, ribozymes of the inventioncan be composed of modified oligonucleotides (e.g., for improvedstability, targeting, etc.). These can be delivered to cells whichexpress the target HSR1 molecules in vivo. A preferred method ofdelivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme tocatalyse HSR1 cleavage. However, because ribozymes, unlike antisensemolecules, are catalytic, a lower intracellular concentration may berequired to achieve an adequate level of efficacy.

Ribozymes can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules, as discussed above. Ribozymetechnology is described further in Intracellular Ribozyme Applications:Principals and Protocols, Rossi and Couture eds., Horizon ScientificPress, 1999.

7. Triple Helix Forming Oligonucleotides (TFOs)

Nucleic acid molecules useful to modulate HSR1 and/or HSF/HSR1/eEF1Afunction or HSR1 gene expression via triple helix formation arepreferably composed of deoxynucleotides. The base composition of theseoligonucleotides is typically designed to promote triple helix formationvia Hoogsteen base pairing rules, which generally require sizeablestretches of either purines or pyrimidines to be present on one strandof a duplex. Nucleotide sequences may be pyrimidine-based, resulting inTAT and CGC triplets across the three associated strands of theresulting triple helix. The pyrimidine-rich molecules provide basecomplementarity to a purine-rich region of a single strand of the duplexin a parallel orientation to that strand. In addition, nucleic acidmolecules may be chosen that are purine-rich, e.g., those containing astretch of G residues. These molecules will form a triple helix with aDNA duplex that is rich in GC pairs, in which the majority of the purineresidues are located on a single strand of the targeted duplex,resulting in GGC triplets across the three strands in the triplex.

Alternatively, sequences can be targeted for triple helix formation bycreating a so-called “switchback” nucleic acid molecule. Switchbackmolecules are synthesized in an alternating 5′-3′, 3′-5′ manner, suchthat they base pair with first one strand of a duplex and then theother, eliminating the necessity for a sizeable stretch of eitherpurines or pyrimidines to be present on one strand of a duplex.

Similarly to HSR1-specific RNAi, antisense oligonucleotides, andribozymes, triple helix molecules of the invention can be prepared byany method known in the art. These include techniques for chemicallysynthesizing oligodeoxyribonucleotides and oligoribonucleotides such as,e.g., solid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules can be generated by in vitro or in vivo transcription of DNAsequences “encoding” the particular RNA molecule. Such DNA sequences canbe incorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.

8. Use of the Antisense Oligonucleotides, RNA interference (RNAi)Molecules, Ribozymes, and Triple Helix Forming Oligonucleotides (TFOs)of the Invention in Developing Novel Cancer Treatments

One of the major problems in cancer treatment today is the resistance oftumor cells to existing therapies. At least part of this resistance isdue to increased synthesis of heat shock proteins (HSPs). As specifiedin the Background Section, supra, HSPs are synthesized in all cells inresponse to adverse conditions such as heat stress. They protect thecell from injury by preventing denaturation of cellular proteins andensuring correct folding of newly synthesized polypeptides. In cancercells, high level of HSPs prevents initiation of apoptosis, orprogrammed cell death, in response to therapeutic treatment. Indeed, asdescribed in Tang et al. (Cell Stress and Chaperones 2005; 10:46-58),HSF and HSP levels are elevated in more highly malignant prostatecarcinoma cells. As the synthesis of HSPs in response to stress iscontrolled by transcription factor HSF, finding a way to inhibit HSFactivation in cancer cells will result in increasing efficiency ofexisting anti-cancer treatments.

The present invention provides a novel way to inhibit HSF activation incancer cells by disclosing for the first time that the activation of HSFin response to heat stress requires at least two additional components:translation elongation factor eEF1A and a novel Heat Shock RNA (HSR1).As disclosed herein, these two components act together to activate HSF.The key role played by the newly discovered HSR1 in this processprovides a rational for new therapeutic agents that would blockexcessive production of HSPs in cancer cells thus rendering them moresusceptible to conventional treatments such as chemotherapy, radiationtherapy, thermal therapy, etc.

Accordingly, in conjunction with the novel HSR1-related nucleic acidsand novel HSF/HSR1/eEF1A ternary complex, the present invention providesnovel anti-cancer agents based on the HSR1-specific antisenseoligonucleotides, RNA interference (RNAi) molecules, ribozymes, andtriple helix forming oligonucleotides (TFOs) as well as methods forusing such agents to treat cancer. The novel anti-cancer agents of thepresent invention can be used in conjunction with existing treatments toimprove their effect by increasing the sensitivity of the cells topro-apoptotic stimuli such as thermo-, chemo-, and radiotherapeutictreatments.

Specifically, as disclosed in Examples Section, infra, HSR1 antisenseoligonucleotides such as phosphothioate derivative of theoligonucleotide 6^(HSR1) (SEQ ID NO: 12; complementary to bases 196-240of hamster HSR1 [SEQ ID NO: 1]) are capable of inhibiting HSF1activation in vivo when transfected in BHK and HeLa cells and renderthem heat sensitive. The data show that this treatment dramaticallyreduces the level of HSP expression and, therefore, promotespro-apoptotic processes that are otherwise blocked by increased HSPexpression.

As disclosed in Examples, infra, to develop effective anti-cancer agentsbased on the HSR1-specific antisense oligonucleotides, RNA interference(RNAi) molecules, ribozymes, and triple helix forming oligonucleotides(TFOs) of the invention, candidate molecules can be introduced in athermotolerant cancer cell line (e.g., breast cancer cell line Bcap37;see Wang, et al., Biochem. Biophys. Res. Commun. 2002; 290:1454-1461)followed by evaluating the HSP expression levels and survival of cellsafter heat shock treatment in comparison to that of untransfected cells.HSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)which show the strongest effect on cell survival and HSP expression canbe further optimized (e.g., by increasing their resistance to nucleases,increasing the efficiency of their targeting to cancer cells, increasingtheir sequence specificity [e.g., by introducing phosphothioate ormorpholino modifications or using LNA], and reducing the size) makingthem even more potent in inhibition of cell survival and inhibition ofHSP expression. The most potent anti-cancer molecules selected in tissueculture experiments can be further tested for their ability to affectthe growth of tumors induced in nude mice (e.g., by injection ofthermotolerant cancer cells) and subjected to heat shock treatment or tochemo- or radiation therapy.

In conjunction with the therapeutics of the invention (e.g., theHSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)),the present invention also provides a method for treating cancer in amammal comprising administering said therapeutics to the mammal. In aspecific embodiment, the mammal is human. In another specificembodiment, the method further comprises subjecting the mammal to atreatment selected from the group consisting of radiation therapy,chemotherapy, thermotherapy, and any combination thereof.

Similarly, the present invention provides a method for increasingsensitivity of a cancer cell to an anti-cancer treatment (e.g.,radiation treatment, chemical treatment, thermal treatment, or anycombination thereof) and thus improving efficiency of such anti-cancertreatment in a mammal comprising administering to the mammal thetherapeutics of the invention. The relative timing ofantisense/RNAi/ribozyme/TFO administration and anti-cancer treatmentwould depend on the delivery mechanism for antisense/RNAi/ribozyme/TFOand on the type of the specific anti-cancer treatment used. Generally,cells may become more sensitive to an anti-cancer treatment as soon asone hour after antisense/RNAi/ribozyme/TFO administration.

Cancers treatable using the methods of the present invention includewithout limitation fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio-sarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, lymphoma, leukemia, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, hepatocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma, among others.

As disclosed above, the anti-cancer compositions of the presentinvention are advantageously used in combination with other treatmentmodalities, including without limitation radiation, chemotherapy, andthermotherapy.

Chemotherapeutic agents used in the methods of the present inventioninclude without limitation taxol, taxotere and other taxoids (e.g., asdisclosed in U.S. Pat. Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957;5,292,921; 5,438,072; 5,587,493; European Patent No. EP 253 738; and PCTPublication Nos. WO 91/17976, WO 93/00928, WO 93/00929, and WO96/01815), cisplatin, carboplatin, (and other platinum intercalatingcompounds), etoposide and etoposide phosphate, bleomycin, mitomycin C,CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, methotrexate,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,nitrosoureas, mitomycin, dacarbazine, procarbizine, campathecins,dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,vincristine, vinorelbine, paclitaxel, docetaxel, calicheamicin, and thelike.

Typical radiation therapy used in the methods of the invention includeswithout limitation radiation at 1-2 Gy.

Also encompassed by the present invention is radiation therapy andchemotherapy via local delivery of radioconjugates andchemotherapeutics, respectively. Directing the cytotoxic exposuredirectly to the tumor itself is a commonly used approach to deliver acytotoxic drug while minimizing the cytotoxic exposure of normaltissues. However, one of the factors which limit the effectiveness ofsuch an approach is incomplete induction of tumor cell death because oflimited dose delivery. Thus, it would be highly desirable toconcurrently use the HSR1-specific therapeutics of the invention toenhance the sensitivity of the tumor cells to the particular cytotoxicagent. Tumor-specific delivery is commonly achieved by conjugating acytotoxic agent (e.g., a toxins (such as ricin) or a radioisotope) to anantibody that preferentially targets the tumor (e.g., anti-CD2 inneuroblastoma or anti-Her2-neu in certain breast carcinomas). Thetargeting may be also done with natural targeting (i.e., withradioactive iodine in the treatment of thyroid carcinoma), physicaltargeting (i.e., administration of a radioisotope to a particular bodycavity), or other targeting protein (e.g., ferritin in hepatocellularcarcinoma).

In addition to combination with conventional cancer therapies such aschemotherapy, radiation therapy, thermotherapy, and surgery (tumorresection), HSR1-targeted therapy of a tumor can be combined with otheranti-tumor therapies, including but by no means limited to suicide genetherapy (i.e., introduction of genes that encode enzymes capable ofconferring to tumor cells sensitivity to chemotherapeutic agents such asthymidine kinase of herpes simplex virus or varicella zoster virus andbacterial cytosine deaminase), anti-oncogene or tumor suppressor genetherapy (e.g., using anti-oncogene molecules including monoclonalantibodies, single chain antibody vectors, antisense oligonucleotideconstructs, ribozymes, immunogenic peptides, etc.), administration oftumor growth inhibitors (e.g., interferon (IFN)-γ, tumor necrosis factor(TNF)-α, TNF-β, and similar cytokines, antagonists of tumor growthfactor (TGF)-β and IL-10, etc.), administration of angiogenesisinhibitors (e.g., fragments of angiogenic proteins that are inhibitory[such as the ATF of urokinase], angiogenesis inhibitory factors [such asangiostatin and endostatin], tissue inhibitors of metalloproteinase,soluble receptors of angiogenic factors [such as the urokinase receptoror FGF/VEGF receptor], molecules which block endothelial cell growthfactor receptors, and Tie-1 or Tie-2 inhibitors), vasoconstrictiveagents (e.g., nitric oxide inhibitors), immune therapies with animmunologically active polypeptide (including immunostimulation, e.g.,in which the active polypeptide is a cytokine, lymphokine, or chemokine[e.g., IL-2, GM-CSF, IL-12, IL-4], and vaccination, in which the activepolypeptide is a tumor specific or tumor associated antigen), and thelike.

9. Use of the Antisense Oligonucleotides, RNA interference (RNAi)Molecules, Ribozymes, and Triple Helix Forming Oligonucleotides (TFOS)of the Invention in Developing Novel Anti-Inflammatory Agents

As specified in the Background Section, supra, HSPs, and HSP70 family inparticular, is considered a part of a protective mechanism againstinflammation (Jattela et al., EMBO J. 1992; 11:3507-3512; Morris et al.,Int. Biochem. Cell Biol. 1995; 27:109-122; Ianaro et al., FEBS Lett.2001; 499:239-244; Van Molle et al., Immunity 2002; 16:685-695; Ianaroet al., Mol. Pharmacol. 2003; 64:85-93; Ianaro et al., FEBS Lett. 2001;499:239-244; Ianaro et al., FEBS Lett. 2001; 508:61-66). It follows,that selective HSF-mediated transcriptional activation of HSP genes maylead to remission of the inflammatory reaction.

As disclosed above, the HSR1-specific nucleic acids of the inventionsuch as, e.g., antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs),can be used to activate HSF and in this way provide a basis fordeveloping novel anti-inflammatory therapeutics.

To develop effective anti-inflammatory agents based on the HSR1-specificantisense oligonucleotides, RNA interference (RNAi) molecules,ribozymes, and triple helix forming oligonucleotides (TFOs) of theinvention, candidate molecules can be first tested in vitro, e.g., bymeasuring TNF-α and IL-β secretion in mononuclear cells of humanperipheral blood or RAW 264.7 cells after their stimulation withlipopolysaccharide (LPS).

Therapeutics which produce the strongest anti-inflammatory effect in invitro assays can be further tested in vivo in various animal models ofinflammation. Examples of the useful animal models include withoutlimitation mouse model of LPS-induced TNF-α secretion (Badger et al., J.Pharmac. Env. Therap. 1996; 279:1453-1461), rat paw edema induced bysubplantar injection of λ-carrageenin (Ianaro et al., Mol. Pharmacol.2003; 64:85-93), mouse pain model produced by injection of an irritant,usually acetic acid, into the peritoneal cavity (Collier et al. Pharmac.Chemother. 1968; 32:295-310; Fukawa et al., J. Pharmacol. Meth. 1980;4:251-259; Schweizer et al., Agents Actions 1988; 23:29-31), etc. Inthese models, the activity of the novel therapeutics in inhibitinginflammation can be determined using various methods known in the art,including without limitation, use of contrast ultrasound in conjunctionwith injection of microbubbles, measurement of inflammatory cytokines(such as TNF-α, IL-1, IFN-γ), measurement of activated immune systemcells (e.g., measurement of the invasion or accumulation in an organ ortissue of proinflammatory lymphoid cells or the presence locally orperipherally of activated pro-inflammatory lymphoid cells recognizing apathogen or an antigen specific to the organ or tissue) as well asobservation (reduction of erythema/redness, reduction of elevatedbody/tissue temperature, reduction of swelling/edema, reduction of painin the affected area, reduction of pruritus or burning sensation,improvement in function of the afflicted organ).

HSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)which show the strongest effect in vitro and in animal models can befurther optimized (e.g., by increasing their resistance to nucleases,increasing the efficiency of their targeting to cells, increasing theirsequence specificity [e.g., by introducing phosphothioate or morpholinomodifications or using LNA], and reducing the size) making them evenmore potent in inhibition of various signs of inflammation andactivation of HSP expression.

In conjunction with the therapeutics of the invention (e.g., theHSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)),the present invention also provides a method for inhibiting aninflammatory reaction in a mammal comprising administering saidtherapeutics to the mammal. In a specific embodiment, the mammal ishuman.

The novel therapeutics of the invention can be used in the prophylaxisas well as in the therapeutic treatment of several disorders (diseasesand other pathological inflammatory conditions) caused by or associatedwith an abnormal or undesirable (excessive, nonregulated, orderegulated) inflammatory immune response involving the production ofinflammatory cytokines or other inflammation mediators, includingwithout limitation TNF-α and IL-1β. They include autoimmune diseasessuch as rheumatoid arthritis, insulin-dependent diabetes mellitus,autoimmune thyroiditis, multiple sclerosis, uveoretinitis, lupuserythematosus, scleroderma; other arthritic conditions having aninflammatory component such as rheumatoid spondylitis, osteoarthritis,septic arthritis and polyarthritis; other inflammatory brain disorders,such as meningitis, Alzheimer's disease, AIDS dementia encephalitis,other inflammatory eye inflammations, such as retinitis; inflammatoryskin disorders, such as, eczema, other dermatites (e.g., atopic,contact), psoriasis, burns induced by UV radiation (sun rays and similarUV sources); inflammatory bowel disease, such as Crohn's disease,ulcerative colitis; asthma; other allergy disorders, such as allergicrhinitis; conditions associated with acute trauma such as cerebralinjury following stroke, heart tissue injury due to myocardial ischemia,lung injury such as that which occurs in adult respiratory distresssyndrome; inflammation accompanying infection, such as sepsis, septicshock, toxic shock syndrome, other inflammatory conditions associatedwith particular organs or tissues, such as nephritis (e.g.,glomerulonephritis), inflamed appendix, gout, inflamed gall bladder,chronic obstructive pulmonary disease, congestive heart failure, Type IIdiabetes, lung fibrosis, vascular disease, such as atherosclerosis andrestenosis; and alloimmunity leading to transplant rejection.

The novel anti-inflammatory agents of the present invention can be usedin conjunction with existing therapeutics such as inhibitors of TNF-α,inhibitors of COX-1/COX-2, inhibitors of IL-1β, etc. In a specificembodiment, the novel therapeutics of the invention are administered incombination with Non-Steroidal Anti-Inflammatory Drugs (NSAIDs).Suitable NSAIDs include, but are not limited to, those which inhibitcyclooxygenase, the enzyme responsible for the biosyntheses of theprostaglandins and certain autocoid inhibitors, including inhibitors ofthe various isoenzymes of cyclooxygenase (including, but not limited to,cyclooxygenase-1 and -2), and as inhibitors of both cyclooxygenase andlipoxygenase relates to NSAID, such as the commercially available NSAIDsaceclofenac, acemetacin, acetaminophen, acetaminosalol, acetyl-salicylicacid, acetyl-salicylic-2-amino-4-picoline-acid, 5-aminoacetylsalicylicacid, alclofenac, aminoprofen, amfenac, ampyrone, ampiroxicam,anileridine, bendazac, benoxaprofen, bermoprofen, α-bisabolol,bromfenac, 5-bromosalicylic acid acetate, bromosaligenin, bucloxic acid,butibufen, carprofen, celecoxib, chromoglycate, cinmetacin, clindanac,clopirac, sodium diclofenac, diflunisal, ditazol, droxicam, enfenamicacid, etodolac, etofenamate, felbinac, fenbufen, fenclozic acid,fendosal, fenoprofen, fentiazac, fepradinol, flufenac, flufenamic acid,flunixin, flunoxaprofen, flurbiprofen, glutametacin, glycol salicylate,ibufenac, ibuprofen, ibuproxam, indomethacin, indoprofen, isofezolac,isoxepac, isoxicam, ketoprofen, ketorolac, lornoxicam, loxoprofen,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, metiazinicacid, mofezolac, montelukast, nabumetone, naproxen, niflumic acid,nimesulide, olsalazine, oxaceprol, oxaprozin, oxyphenbutazone,paracetamol, parsalmide, perisoxal, phenyl-acethyl-salicylate,phenylbutazone, phenylsalicylate, pyrazolac, piroxicam, pirprofen,pranoprofen, protizinic acid, reserveratol, salacetamide, salicylamide,salicylamide-O-acetyl acid, salicylsulphuric acid, salicin,salicylamide, salsalate, sulindac, suprofen, suxibutazone, tamoxifen,tenoxicam, tiaprofenic acid, tiaramide, ticlopridine, tinoridine,tolfenamic acid, tolmetin, tropesin, xenbucin, ximoprofen, zaltoprofen,zomepirac, tomoxiprol, zafirlukast and cyclosporine. Additional NSAIDgenera and particular NSAID compounds are disclosed in U.S. Pat. No.6,297,260 and International Patent Application No. WO 01/87890.

10. Use of the Antisense Oligonucleotides, RNA interference (RNAi)Molecules, Ribozymes, and Triple Helix Forming Oligonucleotides (TFOs)of the Invention in Developing Novel Therapeutics to TreatIschemia/Reperfusion Injury and Neurodegenerative Disorders

As specified in the Background Section, supra, HSPs, and HSP70 family inparticular, is also considered a part of a protective mechanism againstcerebral or cardiac ischemia and neurodegenerative diseases (such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis[ALS], Huntington's disease, spinobulbar muscular atrophy, dentatorubralpallidoluysian atrophy, Kennedy disease, spinocerebellar ataxias, etc.)(Westerheide and Morimoto, J. Biol. Chem. 2005; 280:33097-100; Klettner,Drug News Perspect. 2004; 17:299-306; Pockley, Circulation 2002;105:1012-1017; Hargitai et al., Biochem. Biophys. Res. Commun. 2003;307:689-695; Yenari et al., Ann. Neurol. 1998; 44:584-591; Suzuki etal., J. Mol. Cell. Cardiol. 1998; 6:1129-1136; Warrik et al., Nat.Genet. 1999; 23:425-428; Plumier et al., J. Clin. Invest. 1995;95:1854-1860; Marber et al., ibid., pp. 1446-1456; Radford et al., Proc.Natl. Acad. Sci. USA 1996; 93:2339-2342). It follows, that elevatedactivity of HSF and resulting HSF-mediated transcriptional activation ofHSP genes may lead to inhibition/prevention of ischemia/reperfusioninjury and neurodegenerative disorders. Indeed, as summarized in thereviews by Latchman (Cardiovascular Research 2001; 51:637-646) andKlettner (Drug News Perspect. 2004; 17:299-306), a number of studieshave shown that prior induction of the HSPs by mild stress has aprotective effect against a more severe stress, and overexpression of anindividual HSP in cardiac cells or neurons in culture or in the intacttissue also produces a protective effect.

As disclosed above, the HSR1-specific nucleic acids of the inventionsuch as, e.g., antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs),can be used to activate HSF and in this way provide a basis fordeveloping novel therapeutics to treat ischemia/reperfusion injury andneurodegenerative disorders.

To develop effective therapeutics to treat ischemia/reperfusion injuryand neurodegenerative disorders based on the HSR1-specific antisenseoligonucleotides, RNA interference (RNAi) molecules, ribozymes, andtriple helix forming oligonucleotides (TFOs) of the invention, candidatemolecules can be first tested in vitro, e.g., by testing their abilityto prevent/decrease neuronal cell death (e.g., upon expression ofpolyglutamine-expanded proteins such as mutant huntingtin) orcardiomyocyte cell death (e.g., induced by hydrogen peroxide; see Zou etal., Circulation 2003; 108:3024-3030). Therapeutics which produce thebest effect in in vitro assays can be further tested in vivo in variousanimal models of neurodegenerative diseases (see examples in the sectionbelow) or animal models of ischemia/reperfusion injury such as mousemodel created by transiently ligating the left coronary artery followedby release of suture to allow reperfusion (Harada et al., Circulation1998; 97:315-317; Zou et al., Circulation 2003; 108:3024-3030). In thesemodels, the activity of the novel therapeutics in inhibitingneurodegenerative disorders or ischemia/reperfusion injury can bedetermined using various methods known in the art, including withoutlimitation, determination of electrical activity of neural tissue ormyocardium, ECG, determination of reduction in neuronal or cardiomyocytecell death, etc.

HSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)which show the strongest effect in vitro and in animal models can befurther optimized (e.g., by increasing their resistance to nucleases,increasing the efficiency of their targeting to cells, increasing theirsequence specificity [e.g., by introducing phosphothioate or morpholinomodifications or using LNA], and reducing the size) making them evenmore potent in inhibition of various signs of neurodegeneration orischemia/reperfusion injury and activation of HSP expression.

In conjunction with the therapeutics of the invention (e.g., theHSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)),the present invention also provides a method for inhibiting aneurodegenerative disorder or ischemia/reperfusion injury in a mammalcomprising administering said therapeutics to the mammal. In a specificembodiment, the mammal is human.

The novel therapeutics of the invention can be used in the prophylaxisas well as in the therapeutic treatment of cerebral and cardiacischemia/reperfusion injury as well as various neurodegenerativediseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis [ALS], Huntington's disease, spinobulbar muscularatrophy, dentatorubral pallidoluysian atrophy, Kennedy disease,spinocerebellar ataxias, etc.).

The novel agents of the present invention can be used in conjunctionwith existing treatments such as pharmacotherapy (e.g., therapy usingcholinesterase inhibitors or NMDA receptor antagonists [in cases ofAlzheimer's disease], tissue plasminogen activator (tPA) therapy [incases of stroke], Vascular Endothelial Growth Factor (VEGF) therapy [incases of ischemia]) or mechanical intervention (e.g., bypass surgery,coronary angioplasty followed by stent implantation, etc.).

11. Use of the Antisense Oligonucleotides, RNA interference (RNAi)Molecules, Ribozymes, and Triple Helix Forming Oligonucleotides (TFOs)of the Invention in Developing Novel Anti-Aging Therapeutics

As specified in the Background Section, induction of heat shock eitherby temperature or HSF overexpression can extend life span in modelorganisms. Because the heat shock response is a general protectionmechanism, its therapeutic activation would be therefore useful intreating various age-related diseases (e.g., atherosclerosis andage-related neurodegenerative diseases such as Alzheimer's, Parkinson's,amyotrophic lateral sclerosis [ALS], etc.). Usually, in aged organisms,the general protective systems such as antioxidant protection system andheat shock induction system (both needed to protect damaged proteins)get compromised. One therefore can expect the beneficial role of HSFactivation in age-related diseases. For example, amyloid plaqueformation in Alzheimer's disease (i.e., extracellular aggregates of Aβpeptides which are deposited as amyloid fibrils or amorphous aggregatesdue to aberrant processing of the full-length beta-amyloid precursorprotein (APP)) can be delayed or reversed by overexpression of HSF orHSPs.

As disclosed above, the HSR1-specific nucleic acids of the inventionsuch as, e.g., antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs),can be used to activate HSF and in this way provide a basis fordeveloping novel therapeutics to treat age-related diseases.

To develop effective anti-aging agents based on the HSR1-specificantisense oligonucleotides, RNA interference (RNAi) molecules,ribozymes, and triple helix forming oligonucleotides (TFOs) of theinvention, the ability of candidate molecules to retard or suppressvarious symptoms of age-related diseases can be first tested in vitro incultured cells (e.g., fibroblasts, hepatocytes, motor neurons) and/or inmodel organisms (e.g., C. elegans, Drosophila, mice).

For example, in connection with Alzheimer's disease, neuroblastoma cellscan be used (e.g., SK-N-SH cells, ATCC Accession No. HTB-11, Biedler etal., Cancer Res. 1973; 33:2643-52) as they are known to secretebeta-amyloid precursor protein (APP) derivatives into the conditionedmedium. The levels of these secreted derivatives of APP can be estimatedby probing the conditioned media with specific antibodies to APP using,e.g., the method of Western blotting or ELISA (enzyme linkedimmunosorbent assay). As animal models of Alzheimer's disease, one canuse transgenic mouse animal models expressing APP minigenes that encodeFAD-linked APP mutants (e.g., swe or 717, as disclosed, e.g., in U.S.Pat. No. 5,912,410) or the double mutant mouse model described byBorchelt et al. (Neuron 1997; 19:939-945). The latter transgenic micecoexpress an early-onset familial AD (FAD)-linked human presenilin 1(PS1) variant (A246E) and a chimeric mouse/human APP harboring mutationslinked to Swedish FAD kindreds (APPswe). These mice develop numerousamyloid deposits much earlier than age-matched mice expressing APPsweand wild-type human PS1. Expression of APP minigenes that encodeFAD-linked APP mutants and, in particular, co-expression of the mutanthuman PS1 A246E and APPswe elevates levels of Aβ in the brain, and thesemice develop numerous diffuse Aβ deposits and plaques in the hippocampusand cortex (Calhoun et al., Proc. Natl. Acad. Sci. USA 1999;96:14088-14093). Similarly to humans suffering from AD, these and othertransgenic animal models are characterized by various cognitive defectssuch as loss of neurons, learning deficits, problems in objectrecognition memory, and problems with alternation-spatial reference andworking memory (Chen et al., Nature 2000; 408:975-979).

HSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)which show the strongest effect in vitro and in animal models can befurther optimized (e.g., by increasing their resistance to nucleases,increasing the efficiency of their targeting to cells, increasing theirsequence specificity [e.g., by introducing phosphothioate or morpholinomodifications or using LNA], and reducing the size) making them evenmore potent in activation of HSP expression.

In conjunction with the therapeutics of the invention (e.g., theHSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)),the present invention also provides a method for treating an age-relateddisease in a mammal comprising administering said therapeutics to themammal. In a specific embodiment, the mammal is human.

Age-related diseases that can be treated using HSR1-specific antisenseoligonucleotides, RNA interference (RNAi) molecules, ribozymes, andtriple helix forming oligonucleotides (TFOs) of the present inventioninclude, without limitation, atherosclerosis, neurodegenerativediseases, such as chronic neurodegeneration (e.g., associated withAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis[ALS], etc.), cerebrovascular dementia, acute neurodegeneration (e.g.,associated with stroke and trauma), etc.

The novel anti-aging agents of the present invention can be used inconjunction with existing therapeutics such as statins, non-steroidalanti-inflammatory drugs (NSAIDs), etc.

12. Use of the Antisense Oligonucleotides, RNA interference (RNAi)Molecules, Ribozymes, and Triple Helix Forming Oligonucleotides (TFOs)of the Invention in Developing Novel Anti-Deafness Therapeutics

As disclosed in the Background Section, HSF1-mediated induction of HSPshas been also implicated in protection of sensory hair cells againstacoustic overexposure, hyperthermia and ototoxic drugs. One thereforecan expect the beneficial role of HSF1 activation in treatment ofdeafness and related disorders.

As disclosed above, the HSR1-specific nucleic acids of the inventionsuch as, e.g., antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs),can be used to activate HSF and in this way provide a basis fordeveloping novel therapeutics to treat deafness and related diseases.

To develop effective anti-deafness agents based on the HSR1-specificantisense oligonucleotides, RNA interference (RNAi) molecules,ribozymes, and triple helix forming oligonucleotides (TFOs) of theinvention, the ability of candidate molecules to prevent or amelioratedeafness can be first tested in vitro in cultured cells and/or in modelorganisms (e.g., by monitoring the loss of the sensory hair cells andthe auditory function in mice subjected to acoustic overexposure,hyperthermia or ototoxic drugs.).

HSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)which show the strongest effect in vitro and in animal models can befurther optimized (e.g., by increasing their resistance to nucleases,increasing the efficiency of their targeting to cells, increasing theirsequence specificity [e.g., by introducing phosphothioate or morpholinomodifications or using LNA], and reducing the size) making them evenmore potent in activation of HSP expression.

In conjunction with the therapeutics of the invention (e.g., theHSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)),the present invention also provides a method for treating deafness in amammal comprising administering said therapeutics to the mammal. In aspecific embodiment, the mammal is human. The novel anti-deafness agentsof the present invention can be used in conjunction with existingtherapeutics.

13. Use of the Antisense Oligonucleotides, RNA interference (RNAi)Molecules, Ribozymes, and Triple Helix Forming Oligonucleotides (TFOs)of the Invention in Developing Novel Anti-HIV Therapeutics

As specified in the Background Section, supra, Human ImmunodeficiencyVirus (HIV) LTR suppression can occur under hyperthermic conditions(Gerner et al., Int. J. Hyperthermia 2000; 16:171-181; Steinhart et al.,J. AIDS Hum. Retrovirol. 1996; 11:271-281; Ignatenko and Gerner, Exp.Cell Res. 2003; 288:1-8; see also Brenner and Wainberg, Expert Opin.Biol. Ther. 2001; 1:67-77). It follows, that HSF-mediatedtranscriptional activation may lead to inhibition of HIV transcription.

As disclosed above, the HSR1-specific nucleic acids of the inventionsuch as, e.g., antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs),can be used to activate HSF and in this way provide a basis fordeveloping novel therapeutics to treat HIV infection.

To develop effective anti-HIV agents based on the HSR1-specificantisense oligonucleotides, RNA interference (RNAi) molecules,ribozymes, and triple helix forming oligonucleotides (TFOs) of theinvention, the ability of candidate molecules to mediate LTR suppressioncan be first tested in vitro in reconstituted HIV LTR transcriptionassays or in cultured cells comprising HIV LTR. Therapeutics whichproduce the strongest inhibition of HIV transcription in in vitro assayscan be further tested in vivo in animal models of HIV infection. Inthese models, the activity of the novel therapeutics in inhibiting HIVinfection can be determined using various methods known in the art(e.g., monitoring viral titers or various AIDS-like symptoms).

HSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)which show the strongest effect in vitro and in animal models can befurther optimized (e.g., by increasing their resistance to nucleases,increasing the efficiency of their targeting to cells, increasing theirsequence specificity [e.g., by introducing phosphothioate or morpholinomodifications or using LNA], and reducing the size) making them evenmore potent in inhibition of HIV transcription and activation of HSPexpression.

In conjunction with the therapeutics of the invention (e.g., theHSR1-specific antisense oligonucleotides, RNA interference (RNAi)molecules, ribozymes, and triple helix forming oligonucleotides (TFOs)),the present invention also provides a method for inhibiting HIVtranscription in a mammal comprising administering said therapeutics tothe mammal. In a specific embodiment, the mammal is human.

The novel anti-HIV agents of the present invention can be used inconjunction with existing therapeutics such as azidothimidine (AZT),non-nucleotide analog inhibitors of reverse transcriptase, such asNevirapine (BI-RG-587), TIBO (R82913), pyrinodes (such as R-697,661 andL-696,227), bis(heteroaryl) piperazines (BHAPs, such as U-87201E andU-90,152), atevirdine mesylate (ATV) and R-89431; HIV proteaseinhibitors, including substrate analogs and non-analogs, such as Ro31-8959, A-77003 and A-80987; HIV Tat protein inhibitors, such as Ro5-3335 and Ro 27-7429; blockers of viral entry into cells, such assoluble CD4 protein (sCD4), and chimeric sCD4 derivatives, such asCD4-IgG and CD4-PE40; blockers of HIV RNaseH activity, such as the AZTderivative azidothymidine monophosphate; drugs that alter theintracellular milieu to create conditions less favorable for viralreplication, such as the free-radical scavengers and glutathione-levelrestoring drugs (N-acetylcysteine and similar drugs), thalidomine, etc.

14. Use of the Nucleic Acids of the Invention for GeneratingStress-Resistant Plants

Another object of the present invention is a generation ofstress-resistant plants (i) by generation of plants which aregenetically modified to express or overexpress a constitutively activeHSR1 molecule (e.g., a 3′-truncated human HSR missing 100-150 3′nucleotides such as human HSR1-435 or its plant ortholog) or (ii) bygeneration of plants which are genetically modified so that their normalexpression of an HSR1-encoding gene has been increased or (iii) byadministering to such plants an antisense oligonucleotide or an RNAimolecule or a ribozyme or a TFO that can specifically augment a functionof an endogenous HSR1 or that can specifically activate expression of agene encoding an endogenous HSR1.

The resistance to stresses achieved in such plants includes withoutlimitation various environmental abiotic stresses such asdehydration/extreme water deficiency, unfavorable high or lowtemperatures or abrupt temperature shifts, high salinity, heavy metalstress, acid rain, high light intensities, UV-light, grafting, or thebending of shoots or stems in response to wind and/or rain, or bioticstresses such as pathogen attack.

Preferred plants used for generation of stress-resistant varietiesaccording to the present invention include, without limitation,Arabidopsis and commercially important plants such as sugar storingand/or starch-storing plants, for instance cereal species (rye, barley,oat, wheat, maize, millet, sago etc.), rice, pea, marrow pea, cassaya,sugar cane, sugar beet, potato, tomato, cow pea or arrowroot,fiber-forming plants (e.g. flax, hemp, cotton), oil-storing plants (e.g.rape, sunflower), protein-storing plants (e.g. legumes, cereals,soybeans), vegetable plants (e.g. lettuce, chicory, Brassicaceae speciessuch as cabbage), fruit trees, palms and other trees or wooden plantsbeing of economical value such as in forestry, forage plants (e.g.forage and pasture grasses, such as alfalfa, clover, ryegrass), andornamental plants (e.g. roses, tulips, hyacinths, camellias or shrubs).

Generation of stress-resistant plants according to the present inventioninvolves generation of genetically modified plants prepared byintroducing a polynucleotide into plant cells and regenerating thetransformed cells to plants by methods well known to the person skilledin the art.

Methods for the introduction of foreign genes into plants are also wellknown in the art. These include, for example, the transformation ofplant cells or tissues with T-DNA using Agrobacterium tumefaciens orAgrobacterium rhizogenes, the fusion of protoplasts, direct genetransfer (see, e.g., EP-A 164 575), injection, electroporation, vacuuminfiltration, biolistic methods like particle bombardment,pollen-mediated transformation, plant RNA virus-mediated transformation,liposome-mediated transformation, transformation using wounded orenzyme-degraded immature embryos, or wounded or enzyme-degradedembryogenic callus and other methods known in the art. The vectors usedin the method of the invention may contain further functional elements,for example “left border”- and “right border”-sequences of the T DNA ofAgrobacterium which allow stable integration into the plant genome.Furthermore, methods and vectors are known to the person skilled in theart which permit the generation of marker free transgenic plants, i.e.the selectable or scorable marker gene is lost at a certain stage ofplant development or plant breeding. This can be achieved by, forexample co-transformation (Lvznik, Plant Mol. Biol. 13 (1989), 151-161;Penn, Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems whichutilize enzymes capable of promoting homologous recombination in plants(see, e.g., WO97/08331; Bavley, Plant Mol. Biol. 18 (1992), 353-361);Llovd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet.230 (1991), 170-176; Onouchi, Muck Acids Res. 19 (1991), 6373-6378).Methods for the preparation of appropriate vectors are described by,e.g., Sambrook and Russell (2001), Molecular Cloning: A LaboratoryManual, CSH Press, Cold Spring Harbor, N. Y., USA. Suitable strains ofAgrobacterium tumefaciens and vectors as well as transformation ofAgrobacteria and appropriate growth and selection media are well knownto those skilled in the art and are described in the prior art (GV3101(pMK9ORK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic.Acid Res. 12 (1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989),8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976; Koncz,Specialized vectors for gene tagging and expression studies. In: PlantMolecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.),Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4,1-46; An, EMBO J. 4 (1985), 277-287). Methods for the transformationusing biolistic methods are well known to the person skilled in the art;see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology11 (1993), 1553-1558 and Christou Trends in Plant Science 1 (1996),423-431. Microinjection can be performed as described in Potrvkus andSpangenbern (ads.), Gene Transfer To Plants. Springer Verlag, Berlin,N.Y. (1995). The transformation of most dicotyledonous plants ispossible with the methods described above. But also for thetransformation of monocotyledonous plants several successfultransformation techniques have been developed. These include the;transformation using biolistic methods as, e.g., described above as wellas protoplast transformation, electroporation of partially permeabilizedcells, introduction of DNA using glass fibers, etc. Also, thetransformation of monocotyledonous plants by means ofAgrobacterium-based vectors has been described (Chan, Plant Mol. Biol.22 (1993), 491-506; Hiei, Plant J. 6 (1994) 271-282; Denn, Science inChina 33 (1990), 28-34; Wilmink, Plant Cell Reports 11 (1992), 76-80;May, Bio/Technology 13 (1995), 486-492; Conner and Dormisse, Int. J.Plant Sci. 153 (1992), 550-555; Ritchie, Transgenic Res. 2 (1993),252-265). An alternative system for transforming monocotyledonous plantsis the transformation by the biolistic approach (Wan and Lemaux, PlantPhysiol. 104 (1994), 37-48; Vasii, Bio/Technology 11 (1993), 1553-I1558; Ritala, Plant Mol. Biol. 24 (1994) 317-325; Spencer, Theor. Appl.Genet. 79 (1990), 625-631). The transformation of maize in particularhas been repeatedly described in the literature (see for instance WO95/06128, EP 0 513 849, EP 0 465 875, EP 29 24 35; Fromm, Biotechnology8, (1990), 833-844; Gordon-Kamm, Plant Cell 2, (1990), 603-618; Koziel,Biotechnology 11 (1993), 194-200; Moroc, Theor. Appl. Genet. 80, (1990),721-726). The successful transformation of other types of cereals hasalso been described for instance of barley (Wan and Lemaux, supra;Ritala, supra, Krens, Nature 296 (1982), 72-74), wheat (Nehra, Plant J.5 (1994), 285-297) and rice. For transformation of pasture grass, seeTrinh, Plant Cell Reports 17 (1998), 345-355; Hoffmann, MolecularPlant-Microbe Interactions 10 307-315; Altpeter, Molecular Breeding 6(2000), 519-528; Larkin, Transgenic Research (1996), 325-335, Voise Y,Plant Cell Reports 13 (1994), 309-314 or Tabe, J. Anim. Sci. 73 (1995),2752-2759. For transformation of sugar cane, see Bower and Birch, PlantJournal 2(3) (1992), 409-416. For transformation of sugar beet, see WO91/13159. For transformation of vegetable plants as exemplified fortomato, see Chvi and I Phillips, Plant Cell Reports 6 (1987), 105-108;Davis, Plant Cell, Tissue & Organ! Culture 24, 1991 115-121; van Roekel,Plant Cell Reports 12 (1993), 644-647. For sweet tomato, see Newell,Plant Science 107 (1995), 215-227. For transformation of potato, wheat,barley, rape, soybean, rice, maize, see Excerpts from the text book“Gene Transfer to Plants”, Springer Verlag, Lab Manual, 1995.

The resulting transformed plant cell can then be used to regenerate atransformed plant in a manner known by a skilled person.

15. Screening Methods of the Invention

In one embodiment, the present invention also provides a method foridentifying a candidate compound useful for modulating a function of aeukaryotic Heat Shock RNA (HSR1) and/or HSF/HSR1/eEF1A ternary complex,said method comprising: (a) contacting a first cell with a test compoundfor a time period sufficient to allow the cell to respond to saidcontact with the test compound; (b) determining in the cell prepared instep (a) the function of the HSR1 and/or HSF/HSR1/eEF1A ternary complex;and (c) comparing the function of the HSR1 and/or HSF/HSR1/eEF1A ternarycomplex determined in step (b) to the function of the HSR1 and/orHSF/HSR1/eEF1A ternary complex in a second (control) cell that has notbeen contacted with the test compound; wherein a detectable change inthe function of HSR1 and/or HSF/HSR1/eEF1A ternary complex in the firstcell in response to contact with the test compound compared to thefunction of the HSR1 in the second cell that has not been contacted withthe test compound, indicates that the test compound modulates thefunction of the HSR1 and is a candidate compound. In a specificembodiment, both test and control cells are subjected to stress (e.g.,heat shock). The test compound can be added after cells had beensubjected to stress, or after a preconditioning stress but before thelethal stress, or before cells had been subjected to stress. A functionof HSR1 and/or HSF/HSR1/eEF1A ternary complex assayed according to thismethod can be any function, e.g., stress/temperature-inducedconformational change of HSR1, interaction of HSR1 with HSF, interactionof HSR1 with eEF1A, formation of HSF/HSR1/eEF1A ternary complex,activation of HSF-mediated DNA binding, activation of HSP expression,thermotolerance, etc.

In another embodiment, the present invention provides a method foridentifying a candidate compound capable of binding to a eukaryotic HeatShock RNA (HSR1) or HSF/HSR1/eEF1A ternary complex, said methodcomprising: (a) contacting the HSR1 or HSF1/HSR1/eEF1A ternary complexwith a test compound under conditions that permit binding of the testcompound to the HSR1 or HSF1/HSR1/eEF1A ternary complex; and (b)detecting binding of the test compound to the HSR1 or HSF1/HSR1/eEF1Aternary complex. The binding of the test compound to the HSR1 orHSF1/HSR1/eEF1A ternary complex can be detected, e.g., by detecting HSFDNA binding in crude extracts (using, e.g., electrophoretic mobilityshift assays (EMSA)), HSP expression (using, e.g., immunochemistry), orcell thermotolerance (using, e.g., MTS cell viability assays). In aspecific embodiment, the conditions that permit binding of the testcompound to the HSR1 are stress conditions (e.g., heat shockconditions).

The above-identified screening methods can be used to identify acandidate compound that can be used to generate stress-resistant plantsor to treat a condition that can be treated by modulating a function ofa eukaryotic HSR1 and/or HSF/HSR1/eEF1A ternary complex. Such conditionsinclude cancer, inflammation, ischemia, neurodegeneration, age-relateddiseases, HIV infection, deafness, and related disorders.

Test compounds can be selected without limitation from small inorganicand organic molecules (i.e., those molecules of less than about 2 kDa,and more preferably less than about 1 kDa in molecular weight),polypeptides (including native ligands, antibodies, antibody fragments,and other immunospecific molecules), oligonucleotides, polynucleotides,or a chimera or derivative thereof.

As disclosed in Examples, infra, cellular factors that are associatedwith HSR1 and/or HSF/HSR1/eEF1A complex before and after stress (e.g.,heat shock) in vivo can be identified by co-immunoprecipitation (e.g.,using antibodies against HSR1 (e.g., modified HSR1), eEF1A or HSF) or invivo crosslinking with formaldehyde combined with affinitychromatography using biotinylated HSR1-antisense oligonucleotides as atag.

In addition, compounds that specifically bind to a eukaryotic HSR1and/or HSF/HSR1/eEF1A ternary complex and/or modulate a function of aeukaryotic HSR1 and/or HSF/HSR1/eEF1A ternary complex in vivo can beidentified using yeast “n-hybrid systems”, e.g., yeast one-hybrid systemto detect DNA-protein interactions, yeast two-hybrid system to detectprotein-protein interactions, yeast RNA-based three-hybrid system todetect RNA-protein interactions, and yeast ligand-based three-hybridsystem to detect small molecule-protein interactions, including systemsto detect trimeric interactions, ligand-receptor interactions,interactions that require particular post-translational modifications aswell as “reverse n-hybrid systems” to identify mutations, peptides orsmall molecules that dissociate macromolecular interactions. (for reviewsee Vidal and Legrain, Nucleic Acids Res. 1999; 27:919-29). The use ofvarious types of RNA-based three-hybrid system (SenGupta et al., Proc.Natl. Acad. Sci. USA 1996; 93:8496-501) is particularly preferred toidentify compounds that interact with HSR1 in vivo.

Compounds that specifically bind to a eukaryotic HSR1 and/orHSF/HSR1/eEF1A ternary complex and/or modulate a function of aeukaryotic HSR1 and/or HSF/HSR1/eEF1A ternary complex can be alsoidentified by high-throughput screening (HTS) assays, includingcell-based and cell-free assays, directed against individual targets.Several methods of automated assays that have been developed in recentyears enable the screening of tens of thousands of compounds in a shortperiod of time (see, e.g., U.S. Pat. Nos. 5,585,277, 5,679,582, and6,020,141). Such HTS methods are particularly preferred. One possibleapproach uses recombinant bacteriophage to produce large libraries.Using the “phage method” (Scott and Smith, Science 1990; 249:386-390;Cwirla, et al., Proc. Natl. Acad. Sci. USA 1990; 87:6378-6382; Devlin etal., Science 1990; 49:404-406), very large libraries can be constructed(106-108 chemical entities). A second possible approach uses primarilychemical methods, of which the Geysen method (Geysen et al., MolecularImmunology 1986; 23:709-715; Geysen et al. J. Immunologic Method 1987;102:259-274; and the method of Fodor et al. (Science 1991; 251:767-773)are examples. Furka et al. (14th International Congress of Biochemistry,1988, Volume #5, Abstract FR:013; Furka, Int. J. Peptide Protein Res.1991; 37:487-493), and U.S. Pat. Nos. 4,631,211 and 5,010,175 describemethods to produce a mixture of peptides that can be tested as agonistsor antagonists. In another aspect, synthetic libraries (Needels et al.,Proc. Natl. Acad. Sci. USA 1993; 90:10700-4; Ohlmeyer et al., Proc.Natl. Acad. Sci. USA 1993; 90:10922-10926; PCT Publication Nos. WO92/00252 and WO 94/28028) and the like can be used to screen forcandidate compounds according to the present invention.

16. Genetically Modified Animals

Genetically modified animals can be prepared for studying the biologicalfunction of HSR1 in vivo and for screening and/or testing candidatecompounds for their ability to affect the expression and/or function ofHSR1 or HSF/HSR1/eEF1A ternary complex as potential therapeutics fortreating cancer, inflammation, ischemia, neurodegeneration, age-relateddiseases, HIV infection, deafness, and related disorders.

To investigate the function of HSR1 in vivo in animals, HSR1-specificpolynucleotides of the invention or modulatory HSR1-specific antisensenucleic acids, RNAi (e.g., shRNA or siRNA), ribozymes, or TFOs can beintroduced into test animals, such as mice or rats, using, e.g., viralvectors or naked nucleic acids. Alternatively, transgenic animals can beproduced. Specifically, “knock-in” animals with the endogenous HSR1 genesubstituted with a heterologous gene or an ortholog from another speciesor a mutated HSR1 gene, or “knockout” animals with HSR1 gene partiallyor completely inactivated, or transgenic animals expressing oroverexpressing a wild-type or mutated HSR1 gene (e.g., upon targeted orrandom integration into the genome) can be generated.

HSR1-specific nucleic acids can be introduced into animals using viraldelivery systems. Exemplary viruses for production of delivery vectorsinclude without limitation adenovirus, herpes virus, retroviruses,vaccinia virus, and adeno-associated virus (AAV). See, e.g., Becker etal., Meth. Cell Biol. 1994; 43:161-89; Douglas and Curiel, Science &Medicine 1997; 4:44-53; Yeh and Perricaudet, FASEB J. 1997; 11:615-623;Kuo et al., Blood 1993; 82:845; Markowitz et al., J. Virol. 1988;62:1120; Mann et al., Cell 1983; 33:153; U.S. Pat. Nos. 5,399,346;4,650,764; 4,980,289; 5,124,263; and International Publication No. WO95/07358.

In an alternative method, an HSR1-specific nucleic acid can beintroduced by liposome-mediated transfection, a technique that providescertain practical advantages, including the molecular targeting ofliposomes to specific cells. Directing transfection to particular celltypes (also possible with viral vectors) is particularly advantageous ina tissue with cellular heterogeneity, such as the brain, pancreas,liver, and kidney. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

In another embodiment, target cells can be removed from an animal, and anucleic acid can be introduced as a naked construct. The transformedcells can be then re-implanted into the body of the animal. Nakednucleic acid constructs can be introduced into the desired host cells bymethods known in the art, e.g., transfection, electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, use of a gene gun or use of a DNA vectortransporter. See, e.g., Wu et al., J. Biol. Chem. 1992; 267:963-7; Wu etal., J. Biol. Chem. 1988; 263:14621-4.

In yet another embodiment, HSR1-specific nucleic acids can be introducedinto animals by injecting naked plasmid DNA containing a HSR1-specificnucleic acid sequence into the tail vein of animals, in particularmammals (Zhang et al., Hum. Gen. Ther. 1999; 10:1735-7). This injectiontechnique can also be used to introduce siRNA targeted to HSR1 intoanimals, in particular mammals (Lewis et al., Nature Genetics 2002;32:105-106).

As specified above, transgenic animals can also be generated. Methods ofmaking transgenic animals are well-known in the art (for transgenic micesee Gene Targeting: A Practical Approach, 2nd Ed., Joyner ed., IRL Pressat Oxford University Press, New York, 2000; Manipulating the MouseEmbryo: A Laboratory Manual, Nagy et al. eds., Cold Spring Harbor Press,New York, 2003; Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, Robertson ed., IRL Press at Oxford University Press, 1987;Transgenic Animal Technology: A Laboratory Handbook, Pinkert ed.,Academic Press, New York, 1994; Hogan, Manipulating the Mouse Embryo,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986;Brinster et al., Proc. Nat. Acad. Sci. USA 1985; 82:4438-4442; Capecchi,Science 1989; 244:1288-1292; Joyner et al., Nature 1989; 338:153-156;U.S. Pat. Nos. 4,736,866; 4,870,009; 4,873,191; for particle bombardmentsee U.S. Pat. No. 4,945,050; for transgenic rats see, e.g., Hammer etal., Cell 1990; 63:1099-1112; for non-rodent transgenic mammals andother animals see, e.g., Pursel et al., Science 1989; 244:1281-1288 andSimms et al., Bio/Technology 1988; 6:179-183; and for culturing ofembryonic stem (ES) cells and the subsequent production of transgenicanimals by the introduction of DNA into ES cells using methods such aselectroporation, calcium phosphate/DNA precipitation and directinjection see, e.g., Teratocarcinomas and Embryonic Stem Cells, APractical Approach, Robertson ed., IRL Press, 1987). Clones of thenonhuman transgenic animals can be produced according to availablemethods (see e.g., Wilmut et al., Nature 1997; 385:810-813 andInternational Publications No. WO 97/07668 and WO 97/07669).

17. Examples

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

a. General Methods (i) Cell Culture and HS Treatment

HeLa cells (ATCC Accession No. CCL-2) and BHK-21 cells (ATCC AccessionNo. CRL-1632) were grown at 37° C. in an incubator with 5% CO₂. HeLacells were grown in DMEM containing 10% FBS, 2 mM glutamine andantibiotic/antimycotic cocktail (penicillin/streptomycin/fungizone).BHK-21 cells were maintained in DMEM/F-12 (1:1) mixture supplementedwith 10% NBCS, 2 mM glutamine and the same antibiotic cocktail. Heatshock of cultured cells was performed in a water bath adjusted to 43° C.or 45° C. as indicated in the descriptions of figures and relatedexamples. Monolayer cells grown in screw-cap flasks were tightly closed,sealed with parafilm and submerged in the water bath for indicated timeperiods as indicated in the legends. Corresponding control cells weremaintained in tightly closed flasks in the incubator at 37° C.

(ii) Preparation of the Whole Cell Lysate

After removal of medium, cells were scraped into 3 ml (per 75 cm² flask)of ice-cold PBS and collected by a 5 minute centrifugation at 500×g.Cell pellets were resuspended in ice-cold HEDG buffer (20 mM HEPES-NaOH,pH 7.8, 0.5 mM EDTA, 0.5 mM DTT, 10% glycerol, and protease inhibitorcocktail [Complete™ from Roche, Calif.]) containing 0.42M NaCl at aratio of 100 μl per confluent 75 cm² flask, vortexed briefly, andsubjected to three cycles of freezing in liquid nitrogen/thawing at roomtemperature. The lysate was clarified by a 15 minute centrifugation at25,000×g, divided into small aliquots, flash-frozen and stored at −70°C. Total protein concentration was ˜3-5 mg/ml.

(iii) Protein Expression and Purification

The pGex-2T plasmid (Genbank Accession Nos. A01578 and M21676) carryingmouse HSF1 cDNA fused to N-terminal GST was obtained from Dr. K. Sarge(Sarge et al., Mol. Cell. Biol. 1993; 13:1392-1407). E. coli BL-21 cellswere transformed with pGex-2THSF1, grown to OD₆₀₀˜0.6, and induced with0.1 mM IPTG for 5 hours at 28° C. Cells were collected, resuspended inlysis buffer (20 mM tris-Cl, pH 7.9, 0.3 M NaCl and the proteaseinhibitors cocktail) and sonicated. The lysate was cleared at 35,000×g(20 min) and mixed with 1 ml bed volume of glutathione Sepharose(Amersham) per 1 L of initial bacterial culture (30 min at +4° C.). Thebeads were washed successively with 3×10 bed volumes of lysis buffer,2×10 bed volumes of lysis buffer+5 mM ATP+25 mM MgCl₂, and 3×10 bedvolumes of lysis buffer. For thrombin cleavage of the matrix-boundGST-HSF1, a 50% beads suspension in lysis buffer containing 2.5 mM CaCl₂was incubated with 50 units of thrombin per ml of beads for 12 h at 25°C. on a rotator. Thrombin was inhibited by addition of 0.5 mM PMSF, andthe resin was washed twice with 1 bed volume of lysis buffer. Thefractions were combined and used to prepare HSF1-Sepharose or HSF1activation studies.

eEF1A was purified from rat liver essentially as described byKristensen, et al., Biochem. Biophys. Res. Commun. 1998; 245:810-814.Briefly, rat liver was homogenized in a Polytron homogenizer in 4volumes (v/w) of TEDG buffer (50 mM Tris-HCl, pH 7.9 at 4° C., 0.5 mMEDTA, 0.5 mM DTT, 10% glycerol) containing 50 mM NaCl, centrifuged for30 min at 35,000×g and filtered through 4 layers of gauze. The lysatewas then applied to a Q-Sepharose column equilibrated with TEDG bufferwhose output was connected to the input of CM Sepharose columnequilibrated in the same buffer. After washing off unbound proteins, theQ-Sepharose column was disconnected and the CM Sepharose column wasdeveloped with a linear gradient of 0-0.5 M NaCl in TEDG buffer.Fractions containing eEF1A as determined by SDS-PAGE followed byimmunoblotting and/or Coomassie staining were pooled, dialyzed againstTEDG/50 mM NaCl and applied to a phosphocellulose P11 columnequilibrated with TEDG/50 mM NaCl buffer. A linear gradient of 50-250 mMNaCl in TEDG was applied and fractions were analyzed for the presence ofeEF1A as before. Fractions containing eEF1A were pooled and concentratedby ultrafiltration through a10 kDa MWCO membrane. At this stage, thepurity of the eEF1A preparation was typically 80-90% as determined bySDS-PAGE.

(iv) HSF1 Immobilization on Cross-Linked Sepharose 4B

HSF1 immobilization on cross-linked Sepharose 4B was performed via iminebond formed in a reaction of lysine residue side chains within theprotein with aldehyde groups linked to activated Sepharose. Cross-linkedSepharose CL4B (Sigma-Aldrich, St. Louis, Mo.) was activated byperiodate oxidation. The resin was collected by centrifugation andincubated for 5 hours with two bed volumes of 20 mg/ml sodium periodateat room temperature in the dark. The resin was then washed with 5 bedvolumes of water followed by wash with 3 bed volumes of 100 mM NaBO₃, pH9.0. Then 3 bed volumes of purified HSF1 in 100 mM NaBO₃, pH 9.0 wereadded and the mixture was incubated for 3 hours at room temperature withcontinuous inversion mixing. Typical protein concentration used was0.5-1.5 mg/ml. The resin was sedimented by centrifugation and washedtwice with 100 mM NaBO₃, pH 8.0. The washes were pooled and proteinconcentration was determined to estimate the binding efficiency whichtypically was about 80%. Equal volume of freshly prepared 3 mg/ml sodiumborohydrate in 100 mM sodium borate, pH 8.0 was added to Sepharose,incubated 5 minutes on a rotator, diluted 5-fold with 100 mM NaBO₃, pH8.0. The suspension was centrifuged and the supernatant discarded. Theresin was washed repeatedly with 100 mM NaBO₃, pH 8.0 byresuspension/centrifugation until the pH of the supernatant wasapproximately 8.0 (as checked by pH paper). Removal of non-specificallybound proteins and blocking of excess reactive groups was achieved bythree washes with 0.5 M NaCl in 100 mM ethanolamine, pH 8.0.

(v) Electrophoretic Mobility Shift Assays (EMSA) and ChemicalCross-Linking

EMSA was performed as described by Sarge et al. (Mol. Cell. Biol. 1993;13:1392-1407) with minor modifications. Binding reactions contained10-20 μg protein sample for the whole cell lysate or 10 ng of purifiedrecombinant HSF1, 20 mM HEPES-NaOH, pH 7.9, 100 mM NaCl, 1.5 mM MgCl₂,0.5 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, 1 mg/ml BSA, 2.5 μgpoly(dI-dC), and 1.25 ng of ³²P-end labeled HSE oligonucleotide(5′-GCCTCGAATGTTCGCGAAGTTTCG-3′ (SEQ ID NO: 14)) in a final volume of 40μl. Reactions were incubated for about 20 minutes at room temperature,mixed with 4 μl of 0.02% bromophenol blue in 50% glycerol and loadedonto a 4% PAAG and run in 25 mM Tris, 190 mM glycine, 1 mM EDTA (pH 8.3)at 50 mA until the bromophenol blue reached the bottom of the gel. Thegel was dried and analyzed in a phosphorimager.

Purified HSF1 (10 nM) was incubated with eEF1A and HSR1 at the indicatedconcentrations. The crosslinking reagent EGS (Pierce, Rockford, Ill.)was added to 0.2-0.5 mM in the same buffer used for EMSA for 30 min atroom temperature. Reactions were quenched with 75 mM glycine andprocessed for immunoblotting.

(vi) HSF-Sepharose Pull Down Experiments and Isolation of HSR1

Whole cell lysates were prepared as described above and diluted withHEDG buffer to give the final salt concentration of about 0.21 M NaCl. Abed volume of 10 μl of HSF-Sepharose beads per 75 cm² flask of confluentcells was added and mixed on a rotator at 4° C. for 2-4 hours. Beadswere collected by 5 minute centrifugation at 500×g and washed threetimes with 10 bed volumes of HEDG containing 0.21 M NaCl byresuspension/centrifugation. Elution was performed by incubation of thebeads with one bed volume of HEDG containing 0.21 M NaCl at 43° C. for30 minutes on a rotator. Three successive elutions were performed andthe obtained fractions were analyzed by SDS-PAGE.

To isolate HSR1, eEF1A-containing fractions were pooled, supplementedwith 12.5 mM EDTA and 0.25% SDS and treated with 6,000 units ofproteinase K (Fermentas, Hanover, Md.) for 30 minutes at 50° C. RNA wasthen extracted twice with phenol-chloroform and precipitated withisopropanol.

(vii) Transfection and Cell Viability Assay

The oligos (IDT) used for transfection, 6^(HSR1) (5′-AGT CCT CAC ACC ACTCCA ACG TCT GCT TAT GGC GGC GGA TTC AAC-3′) (SEQ ID NO: 12) andanti-6^(HSR1) (5′-GTT GAA TCC GCC GCC ATA AGC AGA CGT TGG AGT GGT GTGAGG ACT-3′) (SEQ ID NO: 13), were phosphothioate modified at 3′ and 5′ends to increase their stability. In the experiment shown in FIG. 6A,6^(HSR1) oligo or 6^(HSR1)/anti-6^(HSR1) double-stranded oligo (0.56 μMfinal concentration) were used to transfect BHK cells using Superfectreagent (Qiagen, Valencia, Calif.) according to the manufacturer'sinstructions. The day following transfection, cells were heat shockedfor 1 hour at 43° C. and a whole cell lysate was prepared to determineHSF1 activity. In some experiments, cells were allowed to recover at 37°C. overnight after heat shock to allow synthesis of HSPs. Next, a wholecell lysate was prepared and analyzed by immunoblotting using HSP72antibodies (Stressgen, Canada) that specifically recognize only theinducible form of HSP70.

In the experiment shown in FIG. 6B, cells were seeded in 24-well platesat a density of 3−5×10⁴ cells per well. The following day, cells (about75% confluent) were transfected using Oligofectamine reagent(Invitrogen, Carlsbad, Calif.) and the final oligo concentration of 100nM according to the manufacturer's instructions. Transfections wereperformed in groups of four wells. Cell viability was determined usingthe MTS reagent-based CellTiter 96® AQueous Non-Radioactive CellProliferation Assay (Promega, Madison, Wis.). The efficiency oftransfection was estimated by transfecting BHK and HeLa cells withCy5-labeled 6^(HSR1) oligonucleotide (IDT) and counting the percentageof fluorescently labeled cells. Normalization of the survival data forthe efficiency of transfection was done according to the followingtransformation: S_(norm)=100−(100−S)/E, where S_(norm) represents thenormalized percentage of surviving cells, S represents the originalpercentage of surviving cells, and E represents efficiency oftransfection. E was estimated to be 0.3 for BHK and 0.7 for HeLa cells.

(viii) Reporter Plasmid Constructs

A reporter plasmid carrying the Renilla luciferase gene under thecontrol of the human HSP70B promoter was constructed by cloning the 2.3kb HindIII-XhoI fragment of the p173OR vector (Stressgen, Canada) intothe HindIII-XhoI digested pMLuc vector (Novagen, San Diego, Calif.). Theresulting pMLucHSP70 plasmid displayed at least 200- and 100-foldinduction of RLuc activity in HeLa and BHK cells, respectively, underthe HS conditions used (typically, 2 hours at 43° C. followed by 12-16hours of recovery at 37° C.).

(ix) siRNA Experiments

In siRNA experiments, cells were transfected in 12-well plates with amixture of 100 ng pMLucHSP70, 250 ng siRNA construct and 50 ng pSV40bGal(Promega, Madison, Wis.) (as an internal control for the transfectionefficiency) using Effectene reagent (Qiagen, Valencia, Calif.) accordingto manufacturer's instructions. Cells were incubated with thetransfection complexes for 24 hours, heat shocked for 2 hr at 43° C. andallowed to recover at 37° C. for 12-16 hr.

Measurement of RLuc activity was performed in whole cell lysates using aRenilla luciferase activity assay kit (Promega, Madison, Wis.). Briefly,cells were rinsed with ice-cold PBS to remove traces of medium andincubated with 150 μl per well of HEDG buffer containing 0.42 M NaCl and0.25% Triton X-100. Lysates were then clarified by centrifugation at25,000×g for 15 minutes and 4 μl aliquots were used for RLuc activityassays.

The retroviral vector pBNsGFP is a modified derivative of pBabeNeo andcarries the cDNA for enhanced green fluorescent protein (EGFP) asdescribes in Morgenstern and Land (Nucleic Acids Res. 1990; 18:3587-3596). For siRNA experiments, a cassette containing the H1 promoterfollowed by a short hairpin-encoding fragment (siHSR1-224)GATCCCCGGAGTGGTGTGAGGACTACTTCAAGAGAGTAGTCCTCACACCACTCCTTTTT GGAAA (SEQID NO: 16) was excised from pSuper vector (OligoEngine, Inc., Seattle,Wash.) and cloned to replace GFP. Similarly, an antisense expressionconstruct was generated by replacing the GFP cDNA with 604 bp HSR1 cDNAin the antisense orientation in respect to the LTR promoter.VSV-G—pseudotyped virus was produced using the Retro-X packaging system(Clontech, Inc.). Infected BHK and HeLa cells were selected for G418resistance. At least 10³ independent colonies were obtained from eachinfection experiment; these were pooled for further analysis. Transienttransfection with RLuc bGal reporter plasmids, heat shock treatment, andreporter gene activity assays were performed as described above fortransient transfections.

Example 1 Identification of eEF1A as HSF-Associated Factor thatStimulates its Activation

To identify putative auxiliary factors involved in the activation ofHSF1 in the lysate of heat shocked cells, the present inventors lookedat proteins from whole cell lysates retained on covalently immobilizedHSF1. Mouse HSF1 was expressed in E. coli as a fusion protein with GST(glutathione-S-transferase). Induction of GST-HSF1 synthesis wasperformed at 28° C. and care was taken not to exceed an HSFconcentration of 0.5 mg/ml during purification so as to minimizespontaneous trimerization. No trimers were detected in our purifiedrecombinant HSF upon examination by chemical cross-linking (FIG. 1C).Purified HSF1 was covalently attached to Sepharose beads. A polypeptideof approximately 45 kDa was retained on HSF1-Sepharose after incubationwith a lysate from heat shocked, but not from untreated, BHK-21 (FIG.1A) or HeLa cells (FIG. 1B). FIG. 1A shows fractionation of lysates ofheat shocked BHK cells and FIG. 1B shows fractionation of lysates ofheat shocked HeLa cells on HSF1-Sepharose. In FIG. 1A, lane 1 shows awhole cell lysate of heat shocked BHK cells; lane 2 shows thesupernatant after incubation of BHK lysates with HSF1 Sepharose; lanes 3show HSF1 Sepharose beads incubated with the lysates and washed; lanes 4show HSF1-Sepharose beads after three successive rounds of elution at43° C.; lanes 5-7 show proteins released from HSF1-Sepharose aftersuccessive rounds of elution at 43° C. In FIG. 1B, lane 1 showssupernatant after the incubation of the lysate of heat shocked HeLacells with HSF1 Sepharose beads; lane 2 shows proteins bound to HSF1Sepharose after the incubation with the lysate of heat shocked HeLacells and washing; lane 3 shows proteins bound to HSF1 Sepharosefollowing three successive rounds of elution at 43° C.; lanes 4-6 showproteins released from HSF1 Sepharose in three successive rounds ofelution at 43° C. Immunoblotting (FIG. 1C) shows that HSF1 coupled toSepharose was predominantly in monomeric form. Where indicated,cross-linking was performed using 0.5 mM EGS.

The bound polypeptide was identified by MALDI-TOF analysis (performed atNYU Mass Spectrometry Facility) as translation elongation factor eEF1A.Similar results were also obtained with GST-HSF1 fusion proteinimmobilized on glutathione Sepharose beads, demonstrating that chemicalmodifications associated with protein immobilization did not alter theability of HSF1 to interact with eEF1A.

The retention of eEF1A on HSF-Sepharose was temperature-sensitive:incubation at 43° C. caused the release of most of the bound eEF1A (FIG.1A, lanes 5-7 for BHK and FIG. 1B, lanes 4-6 for HeLa cells). No eEF1Abinding was observed if BSA-Sepharose was used instead of HSF-Sepharose.The eEF1A polypeptide could be eluted by incubating the resin at 43° C.,a typical heat shock temperature for mammalian cells.

The eEF1A-containing fraction eluted by heat from HSF-Sepharose wastested to determine whether it has any effect on HSF1 function in vitro.Remarkably, the eEF1A fraction was able to activate endogenous HSF1 inthe lysate of unstressed cells as assayed by electrophoretic gelmobility shift assays (EMSA) (FIG. 2A). An aliquot of a whole celllysate of unstressed BHK cells (20 μg) was incubated in the absence(lane 1, 5) or in the presence (lanes 2-4) of fractions 5-7 from FIG.1A. “43°” in lane 1 indicates that the lysate was heat-shocked for 15min prior to EMSA. Lanes 6-9 contained fraction 5 from FIG. 1A. “HSE”(lanes 6-7) indicates the inclusion of an excess of unlabeled HSEoligonucleotide. “Ab” (lanes 8-9) indicates the presence of a monoclonalanti-HSF1 antibody that causes a supershift (lane 9). Purified HSF1 (10nM) was incubated with eEF1A and HSR1 at the indicated concentrations.The crosslinking reagent EGS (Pierce) was added to 0.2-0.5 mM in thesame buffer used for EMSA for 30 min at room temperature. Reactions werequenched with 75 mM glycine and processed for immunoblotting.

Additionally, this eEF1A fraction induced DNA-binding activity ofrecombinant HSF1 (FIG. 2B). The effect was dose-dependent with respectto both the amount of pure HSF1 (or cell lysate, FIG. 2C) and the eEF1Afraction (FIG. 2B). FIG. 2B shows in vitro activation of recombinantHSF1 by the eEF1A fraction. Increasing amounts of purified recombinantmouse HSF1 were incubated in the absence (−) or presence of the eEF1Afraction and analyzed by EMSA. FIG. 2C shows dose-dependent activationof HSF1 in a lysate of unstressed BHK cells. 20 μg of a lysate ofunstressed (C) or heat shocked (HS) cells were incubated with increasingamounts of the eEF1A fraction (1-10 μl) and subjected to EMSA.

Contrary to previous reports (Guo et al., J. Biol. Chem. 2001;276:45791-45799), heating lysates from unstressed cells in the absenceof the eEF1A fraction did not yield any detectable HSF1 activity (FIG.2A, lane 5). Possible reasons for this discrepancy are discussed below.The induced HSF1 DNA binding activity was specific, since it wassensitive to both an anti-HSF antibody and an excess of unlabeled HSEoligonucleotide (FIG. 2A, lane 6, 7 and 8, 9). Activation of purifiedHSF1 by the eEF1A fraction was accompanied by trimerization of HSF asconfirmed by protein-protein chemical crosslinking (FIG. 2D). FIG. 2Dshows that the eEF1A fraction induces trimerization of purifiedrecombinant HSF1. Purified recombinant mouse HSF1 was incubated in theabsence (−) or presence of the eEF1A containing fraction and thecross-linking reagent EGS. Proteins were then separated by SDS-PAGE,transferred to nitrocellulose membrane, and probed with an anti-HSF1antibody.

The interaction between HSF1 and eEF1A in vivo was confirmed in anexperiment in which eEF1A and HSF1 were co-immunoprecipitated from awhole cell lysate (FIG. 3A). In these experiments, BHK cells were heatshocked for the times indicated (HS) and the whole cell lysates analyzedby immunoprecipitation with an anti-eEF1A antibody followed byimmunoblotting with anti-HSF1. In FIG. 3A, “60R” indicates 60 minrecovery after 60 min HS. Anti-HSF1 immunoblotting revealed a smallamount of HSF1 coprecipitating with eEF1A from unstressed cells (FIG.3A, lanes 1-4). This amount increased steadily upon exposure to heatshock (HS) at 43° C., reaching a plateau within 30 minutes of thetemperature shift (FIG. 3A, lanes 6-7). Recovery at 37° C. for 1 hourresulted in almost complete restoration of the initial low level ofco-precipitated HSF1 (FIG. 3A, lane 8). The apparent inconsistency ofthese results with the observation that eEF1A is eluted fromHSF-Sepharose at 43° C. (FIGS. 1A-B) is explained by the fact that HSF1was coupled to Sepharose in the inactive monomeric form (FIG. 1C),suggesting that the affinity of eEF1A for the HSF1 trimer may be greaterthan its affinity for an HSF1 monomer. The release of HSF1 from theinhibitory multichaperone complex during HS (Zou et al., Cell 1998;94:471-480) could also potentially contribute to the factor's greateravailability for binding to eEF1A. In unstressed cells, most HSF1 wouldbe expected to be associated with HSP90, while most eEF1A would besequestered by the cytoskeleton and translational machinery. Indeed,eEF1A has been implicated in a number of cellular processes besidesprotein biosynthesis: it binds actin filaments, genomic RNA of certainviruses, and some transcription factors (Negrutskii et al. Prog. NucleicAcid. Res. Mol. Biol. 1998; 60:47-78; Izawa et al. Biochem. Biophys.Res. Commun. 2000; 278:72-78; Blackwell et al., J. Virol. 1997;71:6433-6444; Murray et al., J. Cell Biol. 1996; 135:1309-1321). FreeeEF1A may accumulate in the cell under HS conditions due to generaltranslational shut down (Panniers, Biochimie 1994; 76:737-747) and as aresult of cytoskeletal collapse (Welch et al., J. Cell. Biol. 1985;101:1198-1211; Welch et al., Ann. N. Y. Acad. Sci. 1985; 455:57-67).These events would ensure the availability of eEF1A molecules that arecapable of forming a complex with HSF1, and thus link the major cellularperturbations caused by HS and HSF1 activation.

To examine whether cytoskeleton-associated eEF1A was implicated in HSFactivation, we treated cells with cytochalasin before subjecting them toHS. Cytochalasin is an alkaloid that binds to the + end of F-actin andprevents actin polymerization, causing the cytoskeleton to collapse(Cooper, J. Cell Biol. 1987; 105:1473-1478). As shown in FIG. 3B,cytochalasin does not affect HSF1 binding to DNA in the absence of HS(lane 4). However, addition of cytochalasin 1 h prior to exposure ofcells to HS results in a dramatic increase in HSF activity (lanes 2 and3). In these experiments, HeLa cells were incubated with 10 μMcytochalasin (FIG. 3B, lanes 3, 4) and either subjected to 1 hour HS at43° C. (FIG. 3B, lanes 2, 3) or maintained at 37° C. (FIG. 3B, lanes 1,4). Whole cell lysates were prepared and assayed by EMSA. These resultsare consistent with the hypothesis that eEF1A derived from thecollapsing cytoskeleton contributes to HSF activation. At the same time,failure of cytochalasin to promote HSF activation in the absence ofstress suggests that yet another factor is required for the eEF1A/HSFcomplex to become responsive to HS.

Example 2 Isolation of HSR1 RNA and Determination of its Role in HSF1Activation

Initial attempts to activate HSF1 with pure eEF1A isolated from ratliver or HeLa cells were unsuccessful, suggesting that theeEF1A-fraction eluted from HSF-Sepharose contained one or moreadditional unidentified component(s) required for HSF1 activation. SinceeEF1A forms a complex with aminoacyl-tRNA in vivo, this interaction wasevaluated for its role in activating HSF1. As shown in FIG. 4A, HSF1binding to DNA was strongly inhibited in vitro by pre-incubation ofwhole cell BHK lysate with ribonuclease A (RNAseA). The effect wasspecific, since addition of excess tRNA prior to the ribonucleasetreatment protected the HSF activity. The lysate (10 μg of totalprotein) was treated with 500 ng RNase A for 1 hour at 37° C. with orwithout excess tRNA. The amount of HSF1 in each lane was monitored byimmunoblotting as shown in the lower panel of FIG. 4A. Similar resultswere obtained using micrococcal nuclease in the presence of Ca²⁺.

These results prompted the present inventors to search for a specificRNA in the eEF1A fraction that participates in HSF1 activation. eEF1Acontaining fraction was treated with proteinase K in the presence ofSDS, extracted twice with phenol:chloroform mixture and ethanolprecipitated in the presence of glycogen as a carrier. Silver stainingof the resulting preparation that was run on a polyacrylamide denaturinggel (8 M urea), revealed a single band of about 2 kb, demonstrating thatit was not tRNA (FIG. 4B). The band (termed “Heat Shock RNA” or “HSR1”)was sensitive ribonuclease A, but not deoxyribonuclease I (FIG. 4B).FIG. 4B shows the silver stained denaturing 4% PAGE of the RNA isolatedfrom pooled eEF1A containing fractions (left panel). Where indicatedHSR1 samples were treated for 30 min at 25° C. with DNase I (10 U) orRNase A (100 ng) before loading onto the gel (right panel).

Addition of purified HSR1 (but not total RNA) to the lysate ofheat-shocked BHK cells treated sequentially with micrococcal nuclease(MNase) (to remove endogenous HSR1) and EGTA (to inactivate thenuclease) restored the DNA binding activity of HSF1 (FIG. 4C). FIG. 4Cshows EMSA of HSF1 from lysates of heat shocked BHK cells (20 μg) thatwere treated sequentially with MNase, EGTA (to inactivate MNase) (lane1), HSR1 (lane 2) or total RNA (lane 3).

Northern blot analysis showed that HSR1 is constitutively expressed inBHK and HeLa cells and that its level remains unchanged during HS (FIG.4D). The Northern blot analysis of HSR1 expression in BHK and HeLa cellswas performed as follows. Total RNA (10 μg) from either heat shocked(HS) or unstressed (C) BHK or HeLa cells was subjected toelectrophoresis in a denaturing agarose gel, transferred to a membrane,and probed with [³²P]-labeled RNA probe corresponding to region 167-405of HSR1 (SEQ ID NO: 4). An 18S RNA probe was used for normalizationpurposes. The inability of HSR1 to bind to various types of oligo(dT)resin indicates that this RNA is not polyadenylated in vivo, and islikely to be untranslated.

Results similar to those described in Examples 1 and 2 were obtainedwith Drosophila Kc cells. Briefly, incubation of the lysate of heatshocked (1 h at 33° C.) Kc cells with either mouse HSF1 or DrosophilaHSF resulted in binding of eEF1A to the beads. The protein was eluted byheating at 43° C. for 30 min and the resulting fraction contained RNAsimilar in size to HSR1 isolated from BHK and HeLa cells. The RNA wascapable of re-activating HSF in the in vitro system after ribonucleasetreatment, suggesting that the RNA-dependent mechanism of HSF activationis conserved among eukaryotes.

Example 3 Cloning, Expression and Functional Analysis of HSR1

cDNAs of HSR1 were cloned from both HeLa and BHK cells (see Methods),and the coding strand was identified in a functional assay (FIG. 5A). Asshown in the upper panel of FIG. 5A, the sense (T3) and antisense (T7)HSR1s were transcribed in vitro by T3 and T7 RNA polymerases,respectively, from PCR fragments carrying T3 or T7 promoters on eachside of HSR1. The resulting synthetic RNAs were tested in the minimalHSF1 activating system (FIG. 5A, lower panel). For the reconstitutionexperiment shown in FIG. 5A, recombinant mouse HSF1 was used along witheEF1A purified from rat liver (as described above). HSR1 isolated fromheat-shocked BHK or HeLa cells (lanes 7, 8) and HSR1-T3 (lane 10)activated HSF when added together with purified eEF1A. However, neithercomponent alone was capable of activating HSF1 (lanes 2-6). The slightstimulating effect of isolated eEF1A (lane 2) on HSF1 activation resultsfrom the residual amount of co-purified HSR1, and could be eliminated byRNAse A treatment. In FIG. 5A, quantitation of HSF1 activation ispresented as the fold increase relative to a background control. Underthese conditions, about 5% of the total HSF1 was activated.

Crosslinking with ethylene glycol-bis-succinimidylsuccinate (EGS)confirmed that HSR1-T3-mediated activation was accompanied by HSF1trimerization in vitro (FIG. 5B). In these experiments, purifiedrecombinant HSF1 was incubated with eEF1A with (FIG. 5B; lanes 2-4) orwithout (lane 1) HSR1-T3 in the presence (lane 2) or absence (lanes 1,3, 4) of RNase A, followed by cross-linking with indicatedconcentrations of EGS, and immunoblotting using an HSF1 antibody.

In contrast, HSR1-T7 not only failed to induce any significant HSF1binding to DNA (FIG. 5A, lane 9), but also suppressed the activatingeffect of HSR1-T3 (FIG. 5A, lane 11). These data show that HSR1-T3represents the sense strand of HSR1 and that this is sufficient tosupport HSF1 activation.

Notably, the mobility of HSR1-T3 depended on the conditions to which itwas exposed prior to electrophoresis analysis. Both Mg²⁺ and elevatedtemperature caused in vitro synthesized HSR1 to migrate similarly toHSR1 isolated from HS cells (FIG. 5C, compare lanes 2 and 3, 4). Thisconformational change of HSR1 may be associated with triggering HSF1activation, as only the slow-migrating form of HSR1 was retained on HSF1Sepharose (FIG. 4B). To eliminate the possibility of RNA processing as acause of variability in HSR1 mobility, PCR analysis of genomic DNA usingHSR1-specific primers was performed. The product obtained with both BHKand HeLa genomic DNA was about 600 nt long. Sequence comparison of thePCR products from BHK and HeLa cells revealed a high degree of identity,with only a 4 nt difference in the PCR products.

Additional evidence of in vivo complex formation between HSF1, eEF1A andHSR1 was obtained from co-immunoprecipitation experiments (FIG. 5D).Immunoprecipitation was performed using an anti-eEF1A antibody as in theexperiment shown in FIG. 3A, with the exception that the cells were heatshocked for 1 h. RNA was extracted from the precipitate, and HSR1 wasdetected by RT-PCR. Since a greater amount of HSR1 was precipitated fromHS cells compared to a control (lanes 3 and 4, FIG. 5D), more HSR1/eEF1Acomplex is likely to form in cells subjected to stress. On the otherhand, the presence of HSR1 in the precipitate from control cells mayindicate that eEF1A/HSR1 complex is pre-assembled under normalconditions.

Example 4 Suppression of HSF1 Activity by HSR1 AntisenseOligonucleotides

To delineate functional domains within HSR1 that are essential for itsHSF-activating function, a set of 15 overlapping 45-mer antisenseoligonucleotides covering the entire length of the cloned hamster HSR1were synthesized (FIG. 5E). Each oligo was tested in the reconstitutedsystem for its ability to suppress HSF1 activation by HSR1/eEF1A. Theresults of this experiment identified at least two domains in HSR1 thatwere essential for HSF1 activation. Four out of 15 oligonucleotides,1^(HSR1) (SEQ ID NO: 9; complementary to hamster HSR1 [SEQ ID NO: 1] nt1-44), 2^(HSR1) (SEQ ID NO: 10; complementary to hamster HSR1 [SEQ IDNO: 1] nt 40-84), 5^(HSR1) (SEQ ID NO: 11; complementary to hamster HSR1[SEQ ID NO: 1] nt 157-201), 6^(HSR1) (SEQ ID NO: 12; complementary tohamster HSR1 [SEQ ID NO: 1] nt 196-240), spanning 5′-terminal and nt157-240 segments of the cloned hamster HSR1 (SEQ ID NO: 1),respectively, inhibited HSF1 activation by more than 90% in the in vitroreconstituted system (FIG. 5E).

A phosphothioate derivative of the oligonucleotide 6^(HSR1)(complementary to bases 196-240; SEQ ID NO: 12) with the strongestnegative effect on HSF activation in vitro was used to transfect BHK andHeLa cells in order to examine its effect on HSF1 activation in vivo.BHK cells were transfected with 6^(HSR1) or a control double strandedoligo (6^(HSR1)/anti-6^(HSR1)), followed by HS treatment. To assess HSF1activation, a whole cell lysate was prepared immediately following HStreatment and subjected to electrophoretic mobility shift analysis(EMSA) (FIG. 6A). In parallel, we monitored the effect of 6^(HSR1) onHSP72 synthesis in vivo. In this case, BHK cells were allowed to recoverat 37° C. for 16 hours after HS. A whole cell lysate was then preparedand analyzed for HSP72 expression by immunoblotting (lower panel). Asshown in FIG. 6A, 6^(HSR1), but not 6^(HSR1)/anti-6^(HSR1), inhibitedactivation of HSF1 in vivo as well as the production of HSP72 inresponse to HS.

Consistent with these data, the antisense HSR1 oligo also compromisedcell survival after HS (FIG. 6B). To avoid potential toxic effects ofexogenous DNA during transfection, the oligo concentration was decreasedsix-fold (to 100 nM) compared to the previous experiment.Thermotolerance was induced by heat shocking cells at 43° C. for 1 h,followed by a 12 h recovery at 37° C. to allow HSP synthesis and asecond lethal HS challenge (0.5-2 h at 45° C.). Cell viability wasdetermined by the MTS-based assay and normalized to the efficiency oftransfection (see Methods). As shown in FIG. 6B, transfection with6^(HSR1) resulted in more than 80% lethality after 60 min of 45° C. HS,while the control anti-6^(HSR1) oligo did not exhibit any significanteffect on cell viability. Similar results were obtained with HeLa cells.

Independent evidence for a role for HSR1 in the HS response in vivo wasobtained via RNAi experiments. Based on data obtained with antisenseoligos, vectors expressing siRNA against different parts of HSR1(siHSR1) were constructed.

Sequences corresponding to HSR1 are grayed out: (SEQ ID NOS 45-46,respectively): siHSR1-160 (ds 64-mer, cloned into BglII/HindIII digestedpSuper)

siHSR1-224 (ds 64-mer, cloned into BglII/HindIII digested pSuper; thisconstruct corresponds to 6^(HSR1) antisense oligo.) (SEQ ID NOS 16 and47, respectively):

mut160: C11→G (ds 64-mer, cloned into BglII/HindIII digested pSuper)(SEQ ID NOS 48-49, respectively):

mut224: G4→C (ds 64-mer, cloned into BglII/HindIII digested pSuper) (SEQID NOS 50-51, respectively):

Both siHSR1-160 and siHSR1-224 were assembled by annealing tocorresponding single-stranded oligos. The resulting double-strandedoligo with BglII-compatible/HindIII overhangs was ligated intoBglII/HindIII digested pSuper vector (OligoEngine, Inc., Seattle,Wash.). Upon transcription these fragments form hairpin structure with19 nt double-stranded stem and 9 nt bubble (TTCAAGAGA (SEQ ID NO: 26)).This hairpin is cleaved in the bubble in the cells to produce dsRNA withUU overhangs on both ends.

The effect of siRNA was monitored by the HS induced expression of aplasmid-derived Renilla luciferase (RLuc) reporter fused to theinducible human hsp70 promoter (see Methods). This construct wasco-transfected with siRNA vectors into HeLa cells and the induction ofRLuc activity by HS was measured. As shown in FIG. 6C, while the RLucactivity was induced about 200-fold by HS treatment followed by recoveryat 37° C., siRNA corresponding to the 6^(HSR1) antisense oligo(siHSR1-224; SEQ ID NO: 20 and SEQ ID NO: 28) strongly inhibited the HSinduction of RLuc. Importantly, a mutant construct carrying a single G→Csubstitution in the siRNA sequence (mut224: G4→C; SEQ ID NO: 21 and SEQID NO: 30) had a significantly diminished effect on RLuc induction byHS.

Finally, HeLa cell lines stably expressing siHSR1 were generated. Thesecell lines were transiently transfected with the RLuc reporter plasmidand the HS induction of RLuc activity was monitored. In agreement withprevious data, cells expressing siHSR1 or HSR1 antisense (aHSR1, SEQ IDNO: 3) but not GFP were deficient in their ability to induce RLucactivity after 2 h HS at 43° C. followed by overnight recovery at 37° C.(FIG. 6D). Moreover, the induction of HSF1 DNA binding activity by HSwas severely impaired in siHSR1, but not in GFP expressing cells (FIG.6D, inset). Predictably, cells stably expressing siHSR1 failed toacquire thermotolerance after HS pre-conditioning (FIG. 6E). Takingtogether, these data show that HSR1 is essential for the HS response inmammalian cells.

Discussion

Recent studies have implicated various untranslated RNAs in asurprisingly wide spectrum of regulatory functions (Nudler and Mironov,Trends Biochem. Sci. 2004; 29:11-17 and Szymanski and Barciszewski, Int.Rev. Cytol. 2003; 231:197-258). The present invention encompasses anovel presumably untranslated RNA that is involved in activation of theHS response. HSR1 likely serves as a cellular thermosensor that assumesan HSF-1 activating conformation in response to elevated temperature.The ability of HSR1 to dramatically change its mobility apparently dueto oligomerization (FIG. 5C) is consistent with this notion. As themaster activator of HSP genes, HSF1, requires two components in order toacquire its DNA binding activity—eEF1A and thermosensor RNA (HSR1). HSR1and/or eEF1A may participate in HSF1 interaction with other componentsof transcription machinery. This model provides a framework fordesigning new potential therapeutics to control heat shock response invivo for cancer treatment and other pharmacological applications.

Example 6 Identification of Genomic Sequences Encoding Human, Drosophilaand Arabidopsis HSR1 and Comparative Sequence and Functional Analysis ofthese Orthologs

A. Identification of Human Genomic HSR1 Sequence and HSR1 Sequences ofDrosophila and Arabidopsis

Human genomic HSR1 sequence was identified as follows. Southern blottingof HeLa genomic DNA digested with PstI was performed using humanHSR1-specific probe (i.e., 562-nt HSR PCR fragment having SEQ ID NO: 2obtained using HSR1 cloned into pBluescript SK vector as a template).The 3.5-5.0 kb PstI fragments, corresponding to the positive HSR1 signalon the Southern blot, were extracted from agarose gel and cloned intopGM3 vector (Promega). The resulting plasmids were transformed intoDH10B cells (Invitrogen) with high efficiency (˜5×10⁸) and lowbackground (less than 2%). About 2×10⁵ transformants were plated,incubated until the colonies reached 0.5-1 mm in size, transferred onnylon membranes and screened by hybridization with radioactive labeledhuman HSR1-specific probe (i.e., 562-nt HSR PCR fragment having SEQ IDNO: 2). 18 positive signals were found. A recombinant plasmid from oneof them was used for sequencing. The resulting sequence (SEQ ID NO: 32)contained the sequence encoding human HSR1 (SEQ ID NO: 37) surrounded onboth 5′ and 3′ end by flanking inverted repeats.

Drosophila HSR1 sequence was identified by PCR from Drosophila genomicDNA using the following reagents and conditions:

PCR reaction mix: 10 X PCR buffer (Fermentas) 10 μL 10 mM dNTPs(Fermentas or Roche) 1 μL Taq polymerase (Fermentas) 0.5 μL 25 mM MgCl₂6 μL genomic DNA (Bio-chain Inst., Inc.) 1-5 μg 100 μM Forward primer 1μL 100 μM Reverse primer 1 μL Water up to 100 μL 1-199 fragment Forwardprimer (HSR1-18): (SEQ ID NO: 38) CCGTCCAATTGAGGTCCG Reverse primer(antiHSR199-183): (SEQ ID NO: 39) CAACCGGTCGATGCAAC 195-378 fragmentForward primer (T7HSR195-209): (SEQ ID NO: 40)GAATTAATACGACTCACTATAGGGTTGAATCCGCCGC Reverse primer (antiHSR378-358):(SEQ ID NO: 41) GTTAGATCATGAACCCGGACG 363-604 fragment Forward primer(T7HSR363-384): (SEQ ID NO: 42)GAATTAATACGACTCACTATAGGGTTCATGATCTAACTCGTTG Reverse primer(antiHSR604-586): (SEQ ID NO: 43) GCCCTTGACTCCAGTCGAC PCR cyclingconditions: 1. 95° C. for 3 min 2. 95° C. for 1 min 3. 55° C. for 45 sec4. 72° C. for 1 min 5. 30-35 cycles of 2-4 step 6. 72° C. for 5 min 7. 4° C.

Comparison of the resulting HSR1 from Drosophila (SEQ ID NO: 33) tohuman HSR1 (SEQ ID NO: 37) using BLAST bl2seq program reveals 98%identity with only 8 substitutions (FIG. 8). The present inventorshypothesized that at least some of the nucleotides corresponding tothese substitutions determine a temperature threshold for the heat shockresponse, because the heat shock response in Drosophila cells is set fora much lower temperature (30-33° C.) as compared to human cells (39-40°C.). The in vitro results described in Example 6D and shown in FIG. 9C(see a more detailed discussion below) demonstrate that this is thecase. Drosophila HSR1 and human HSR1 activate HSF1 at temperaturescorresponding to Drosophila and human heat shock thresholds,respectively. These results supports the notion that HSR1 is a universalRNA thermosensor and identifies the amino acid positions wheresubstitutions in HSR sequence can be made for shifting the threshold forthe heat shock response.

Arabidopsis HSR1 sequence was identified by PCR from genomic DNA usingprimers corresponding to highly conserved distal ends of human HSR1inverted repeats. Specifically, a single primer:5′-TTCTCCAGCTCCAGCAGC-3′ (SEQ ID NO: 44) was used for PCR with ExTaqpolymerase (Takara) from a commercially available Arabidopsis genomicDNA (Promega). The PCR conditions were: (98° C. 15 sec; 56° C. 15 sec;72° C. 35 sec)×45 cycles. The sequence of the resulting ˜1 kb PCRfragment turned out to have very high homology (93% and 80%,respectively, as determined by BLAST bl2seq) to two fragments (each over100 bp) of human HSR1 (HeLa) as shown below:

The sequence homologous to human HSR1 was surrounded by unique flankingsequences, which were, in turn, surrounded, by inverted repeats.

To verify that the Arabidopsis HSR1 obtained by PCR represents acomplete and only HSR1 sequence present in Arabidopsis genome, a libraryscreening is performed using the same method as described above for theidentification of the human genomic HSR1 sequence. Specifically,Southern blotting of Arabidopsis genomic DNA digested with PstI isperformed using Arabidopsis HSR1-specific probe. The PstI fragments,corresponding to the positive HSR1 signal on the Southern blot, areextracted from agarose gel and cloned into pGM3 vector (Promega). Theresulting plasmids are transformed into DH10B cells (Invitrogen) withhigh efficiency and low background. Transformants are plated, incubateduntil the colonies reach 0.5-1 mm in size, transferred on nylonmembranes and screened by hybridization with radioactive labeledArabidopsis HSR1-specific probe. Recombinant plasmids isolated from theclones producing positive signals are used for sequencing.

B. Generation of a Constitutively Active HSR1 Molecule

Based on mfold prediction (seehttp://bioweb.pasteur.fr/seqanal/interfaces/mfold.html), the presentinventors hypothesized that the 3′ 100-150 nt segment of human HSR1plays a self-inhibitory role. It was further hypothesized that, duringtemperature up-shift, base pairing between the 3′-terminal portion ofHSR1 and internal sequences melts, allowing the HSR1 to adopt adifferent (active) conformation, which allows for HSF activation. Toconfirm this prediction, various 3′ truncated human HSR1 molecules weregenerated (HSR1-435; HSR1-474 and HSR1-535). Indeed, the deletion of the3′-terminal 100-150 nt rendered human HSR1 constitutively active, i.e.,active in terms of HSF1 activation regardless of the temperature. Asshown in FIG. 9A, in contrast to the full-length human HSR1 (wt HSR1),3′-truncated human HSR1 (human HSR1-435; SEQ ID NO: 35) was able toactivate HSF1 DNA binding at room temperature (23° C.). Full-lengthhuman HSR1 activated HSF1 (as assessed by HSF1 trimerization and DNAbinding) only at higher temperatures (e.g., 43° C.), unless provided inhigh concentrations (above 100 nM).

Experiments shown in FIG. 9A were performed as follows.

eEF1A was purified from S-100 lysate of HeLa cells obtained as describedin Dignam et al., Nucleic Acids Res. 1983, 11(5):1475-89. 50 ml of S-100lysate was applied to 40 ml DEAE-Sepharose column (GE Healthcare; ˜40 mlbed volume) equilibrated with buffer A (25 mM Tris-HCl, pH 7.5, 1 mMDTT, 0.1 mM EDTA, 25% (v/v) glycerol) containing 50 mM KCl. Theflow-through, which contained eEF-1A, was collected and applied to 15 mlphosphocellulose P11 column (Whatman; ˜15 ml bed volume) equilibratedwith buffer B (25 mM Tris-HCl, pH 7.9, 1 mM DTT, 0.1 mM EDTA, 10% (v/v)glycerol) containing 50 mM KCl. The column was then washed with 3volumes of the same buffer and linear gradient of 50-650 mM KCl (10column volumes) in buffer B was applied and 3 ml fractions werecollected. The fractions were assayed by running a 20 μl aliquot on a10% SDS-PAGE and staining the gel with Coomassie Blue R-250. Fractionscontaining eEF-1A were pooled and concentrated using Centricon orsimilar ultrafiltration device with the 10 kDa MWCO membrane to ˜2 mland stored at −80° C.

Electrophoresis mobility shift assay (EMSA) reactions were performed inthe final volume of 25 μl in a buffer containing 20 mM HEPES-NaOH (pH7.9), 100 mM NaCl, 2 mM DTT (except where indicated), 4 mM MgCl2, and37.5 ng/μl poly(dI-dC) (EMSA buffer). Reactions contained 0.1-10 nMtruncated human HSR1-435 or full-length human HSR1 (prepared by in vitrotranscription with T7 RNA polymerase using PCR fragment as a template),1 nM recombinant mouse HSF1 (prepared as described in the generalMethods section, above), and 20 nM eEF1A. The reactions were assembledon ice by mixing water, EMSA buffer, and RNA. The reactions were thenincubated at the desired temperature in a PCR thermocycler for 15 min,supplemented with eEF1A at 20 nM final and HSF1 at 1 nM final, mixed bypipetting up and down and incubated at 23° C. for 30 min. Threemicroliters of the mix (1:5) of 2.5 ng/μl ³²P-labeled HSEoligonucleotide (5′-GCCTCGAATGTTCGCGAAGTTTCG-3′; SEQ ID NO: 14 (IDTDna)) and loading dye (25% glycerol, 0.02% bromophenol blue) were addedand the incubation continued for another 20 min at 23° C. The reactionswere then loaded on 4% polyacrylamide gel (25 mM tris, 190 mM glycine,pH 8.3—TG buffer) and run for 3 h at 20 mA in TG buffer. The gel wasthen dried and exposed to Phosphoimager Imaging Screen.

FIG. 9A shows EMSA analysis of HSF1 activation in vitro by HSR1-435deletion mutant as compared to the full-length human HSR1. HSF1activation reactions were set up as described above with theconcentration of HSR1-435 and HSR1 equal to 0.1, 1 and 10 nM in eachtriplet and were incubated for 15 min at 23° C. or 43° C. Then HSF1 andeEF1A were added and the reactions were processed as described above. Asshown in the figure, HSF1 is activated in the presence of HSR1-435deletion mutant regardless of the incubation temperature in adose-dependent manner. In contrast, in the presence of the full lengthHSR1, the activation of HSF1 only occurs when the RNA is pre-exposed toelevated temperature (43° C.).

These data show that HSR1-435 deletion mutant is capable of activatingHSF1 in the absence of heat shock, presumably because it is alreadypresent in the active conformation. The result also suggests that 3′part of HSR1 is necessary for the maintaining of inactive conformationunder normal conditions. Thus, a constitutively active human HSR1 can begenerated by deleting the 3′-terminal 100-150 nt. Such constitutivelyactive human HSR1 and its corresponding homologs in other species couldpotentially activate the stress response without actual stress in vivoand could be therefore used for generating cells and trangenic organisms(e.g., transgenic plants) with increased resistance to stress.

C. Testing the Effect of Cys Residues on HSF1 Activation

Two Cys residues in HSF1 were shown to be essential for the activationof the factor in vivo (Ahn and Thiele, Genes Dev. 2003, 17(4):516-28).The effect of Cys residues on HSF1 activation was therefore tested invitro by EMSA analysis in the presence or absence of DTT. The results ofthe test are shown in FIG. 9B. HSF1 activation reactions were set up asdescribed in section B, above, either in the absence (empty circles inFIG. 9B) or in the presence (filled circles in FIG. 9B) of DTT (2 mM)and HSR1 (1 nM). The reactions were then incubated at 37° C. or at 43°C. (as indicated below the gel in FIG. 9B) for 15 min.

As shown in FIG. 9B, HSF1 activation does not occur in the absence ofDTT. In the presence of 2 mM DTT, HSF1 is activated if HSR1 ispre-incubated at 43° C. prior to the addition of HSF1 and eEF1A. Itfollows that DTT is required for the activation of HSF1 in vitro byeEF1A and HSR1 indicating the involvement of Cys residues in thisprocess.

D. Testing Functional Interchangeability of HSR1 Orthologs

To further assess the extent of the functional conservation of the keycomponents of the HSF activating complex, HSR1 isolated from human andDrosophila were used to activate HSF1-eEF1A in in vitro EMSA assays.

Specifically, 1 nM human HSR1 was incubated at the followingtemperatures for 15 min: 23.1° C., 30.8° C., 36.4° C., 39° C., 43.9° C.before the addition of HSF1 and eEF1A and processing the samples asdescribed in section B, above. 1 nM Drosophila HSR1 was incubated at thefollowing temperatures for 15 min: 22.9° C., 24.1° C., 25.9° C., 28.2°C., 30.8° C., 33.6° C., 36.4° C., 39° C., 41.2° C. before the additionof HSF1 and eEF1A and processing the samples as described in section B,above. As shown in FIG. 9C, human HSR1 starts to activate HSF1 atapproximately 36° C. and the activation reaches the maximum atapproximately 43° C., consistent with the mammalian heat shock thresholdtemperature in vivo. Drosophila HSR1 starts to activate HSF1 atapproximately 30° C. and the maximum of activation is reached atapproximately 36° C.

As follows from these data, the threshold temperature of HSF1 activationin in vitro system is controlled by HSR1 making HSR1 a bona fidethermosensor. The temperature-induced conformational transition in HSR1is likely translated into HSF1-activating signal, presumably througheEF1A as an effector protein. The in vitro results shown in FIG. 9C alsodemonstrate that Drosophila HSR1 and human HSR1 are interchangeable intheir ability to activate HSF1 at temperatures corresponding toDrosophila and human heat shock thresholds, respectively.

E. Additional Screening for Functionally Important Domains in HSR1

Additional screening for functionally important domains in HSR1 isperformed using deletion and mutational analysis. In parallel, using aseries of deletions in the HSR1, the minimal portion of HSR1 that stillsupports activation of HSF1 in vitro is identified.

To further delineate the most conserved and functionally importantdomains of HSR1, in addition to direct sequence comparison of human,Drosophila and Arabidopsis HSR1, a comparative analysis of theirsecondary/tertiary structures is performed using RNA nuclease mappingand RNA self-cleavage approach (see Example 7, infra). The cleavagepatterns from RNase and “self cleavage” experiments are compared betweenhuman, Drosophila and Arabidopsis HSR1 and computationally analyzed tofind the common structural domains.

Example 7 Evaluation of HSR1 as a Thermosensor

Based on the experiments in Example 6(D), above, it is hypothesized thatHSR1 serves as a thermosensor that undergoes a conformational change inthe cell in response to elevated temperature. Such a change induces HSFtrimerization and DNA binding. RNA thermosensors have been described inbacteria, although their mode of action must be principally differentfrom that of HSR1 (Storz, Genes Dev. 1999; 13:633-636).

To further determine whether HSR1s sense heat, the structures of invitro transcribed human, Drosophila and Arabidopsis HSR1 are probedusing enzymatic and spontaneous cleavage approaches to test whether RNAundergoes conformational changes upon exposure to a certain elevatedtemperature. Nuclease mapping strategy utilizes several sequence- andstructure-specific nuclease enzymes: mung beans nuclease (cleaves singlestranded RNA (ssRNA)), RNase A (pyrimidine-specific nuclease, cleavesssRNA), RNase TI (cleaves ssRNA 3′ to G), V1 (cleaves double stranded(dsRNA)). Terminally [³²P] labeled HSR1 is treated by nucleases undersingle hit conditions before and after incubation for 30 min at 43° C.(for human HSR1) or 32° C. (for Drosophila HSR1) or 38° C. (forArabidopsis HSR1) and analyzed by denaturing PAGE. The change incleavage pattern indicates change in the secondary structure of HSR1associated with elevated temperature.

Additional structural information is obtained using RNA spontaneous‘self-cleavage’ approach (Soukup, et al., RNA. 1999a; 5:1308-1325). Thisstructure-probing process relies on the inherent chemical instability ofRNA under physiological conditions that occurs primarily due to thespontaneous cleavage of phosphodiester linkages via intramoleculartransesterification reactions. The internucleotide linkages inunstructured regions are more likely to undergo spontaneous cleavagecompared to linkages that reside in highly structured regions of RNA(Soukup, et al., RNA. 1999b; 5:1308-1325).

Example 8 Investigation of Protein-RNA Contacts In HSF1/HSR1/EEF1ATernary Complex Before and after Heat Shock

As the right in vivo conformation of HSR1 may be assumed only in complexwith proteins, the results obtained in RNase mapping and self-cleavageexperiments in Example 7 are used as a starting point for a detailedinvestigation of the structure of HSF1/HSR1/eEF1A ternary complex andthe temperature-inducible conformational changes in HSR1. To addressthis question, the regions of HSF1 and eEF1A are mapped relative to HSR1RNA by chemical derivatization of HSF and eEF1A in the ternary complexusing a variety of highly specific and inducible cross-linkable reagentsincorporated into defined positions in HSR1. The cross-linking isfollowed by mapping adducts using chemical and enzymatic degradation ofderivatized proteins. The following cross-linkable NTP substrates areused: a) 4-thio-UTP, 6-thio-GTP, and 2-Iodo-UTP as short arm (<1 Å)photoactivable cross-linkers (these reagents are commercially availableas monophosphates, e.g., from TriLink Biotechnologies, San Diego,Calif.) and were converted to the triphosphate form as disclosed inGusarov and Nudler, Cell 2001; 107:437-49); b) aryl azido derivative ofthe 5-aminoallyl UTP as a medium arm (˜7 Å) photoactivable cross-linker,and bis(2-iodoethyl)amino derivative of aminoallyl UTP as a long arm(˜12 Å) chemically activable cross-linker (synthesized and used asdisclosed in Nudler, et al., Science 1998; 281:424-428).

In each experiment the cross-linkable reagent is incorporated into asingle defined position in HSR1 using walking technology developed bythe inventors and co-workers, i.e., step-wise transcription in solidphase as disclosed in Nudler, et al., Science 1998c; 281:424-428;Gusarov, et al., Mol. Cell. 1999; 3:495-504; Nudler, et al., Science1994; 265:793-796. This method allows introduction of a radioactivelabel or cross-linking derivative of nucleotides into any desiredposition along the RNA sequence. The principle behind the solid-phasewalking is that the initial elongation complex immobilized onto a solidsupport undergoes rounds of washing (to remove the unincorporated NTPsubstrates) followed by addition of the incomplete set of NTPs (three orless) that allows transcription to proceed to the next DNA positioncorresponding to the first missing NTP (FIGS. 7A-B). Briefly, full sizeHSR1 is cloned so that its sequence starts after position+11 of T7A1promoter. His₆-tagged (SEQ ID NO: 56) E. coli RNA polymerase isimmobilized on Ni⁺⁺ or Co⁺⁺ chelating beads and the transcription isinitiated by addition of T7A1 promoter DNA fused to HSR1 sequence. Afterinitiation of transcription with CpApUpC RNA primer, ATP and GTP, thebeads containing initial elongation complex are washed withtranscription buffer to remove unincorporated NTPs. In the next step(s),the appropriate limited mixture of NTPs is added to move RNA polymeraseto the next desired position (FIG. 7A). At a certain step, thecross-linkable NTP analog and/or radiolabeled NTP is added so that asingle modification is introduced exactly at the desired positionfollowed by chase reaction to complete the RNA synthesis.

Modified or labeled nucleotides can be introduced at any step during thewalking procedure (Nudler et al., Methods Enzymol. 2003; 371:160-9),enabling synthesis of the HSR1 transcript that carries a cross-linkableanalog or radioactive label virtually at any desired position. However,the walking procedure is mostly useful for obtaining RNA modificationsclose to the promoter because of inevitable loss of the material duringmultiple washing steps. To introduce radioactive label or modified basedeep inside the long RNA sequence, a “roadblock” modification of theprocedure is used (FIG. 7B). The latter relies on the mutant form ofEcoRI restriction endonuclease, EcoRQ111 (provided by Dr. Paul Modrich,Duke University; Wright et al., 1989, J. Biol. Chem., 264:11816-11821),which binds EcoRI site but does not cut the DNA. RNA polymerase stopsupon encountering DNA bound EcoRQ111. After washing out unincorporatedNTPs, the “roadblock” is removed by high salt wash, which leaveselongation complex intact (Nudler et al., Cell 1995; 81:351-7; Nudler etal., Methods Enzymol. 2003; 371:160-9). From this point, elongationcomplex is walked to the desired position as described above. Therefore,introducing EcoRI site near the target position in HSR1 sequence by PCRmutagenesis allows incorporating NTP derivatives (e.g., 4-thiouridine or6-thioguanosine) at any distal position along the HSR1 sequence.

The HSR1 derivatives synthesized in vitro using “walking” technique arethen incubated with HSF and/or eEF1A and the cross-linking is initiatedby brief UV irradiation. Following cross-linking, the labeled protein isdigested under the single-hit conditions with cyanogen bromide (CNBr)[cleaves after methionine (M)]. The RNA is digested with ribonucleaseand the reaction mixture is then resolved on SDS-PAGE. This procedureresults in the transfer of radioactive label, which was incorporatedadjacent to the cross-linking derivative, to the protein. Therefore,cross-linking sites on proteins are mapped using limited single-hitprotein degradation with different reagents. The following chemical andenzymatic degradation agents are used:

Agent Cleaves at cyanogene bromide (CNBr) Methionine2-nitro-5-thiocyanobenzoic acid (NTCBA) Cysteine chloro-succinimideTryptophan endoproteinase Lys-C Lysine endoproteinase Glu-C Glutamicacid

Using a combination of these cleaving agents, it is possible to mapcrosslinking sites within 10-20 amino acids (Nudler, et al., Science1998b; 281:424-428). Human and mouse HSF1 contain 12 Met and 5 and 4 Cysresidues, respectively, which makes them well suited for the limiteddegradation analysis. Drosophila HSF contains 26 Met and only 1 Cysresidue but also has 3 Trp residues. Finally, eEF1A sequence includes 12Met and 6 Cys residues. Thus, the limited CNBr and NTCBA degradationyields characteristic peptide patterns for each protein and allowshigh-resolution mapping of cross-linking sites within HSF and eEF1Amolecules.

In addition to crosslinking, chemical footprinting of both RNA andproteins is used to analyze the HSF/eEF1A/HSR1 ternary complex. Thedynamic change of protection areas on RNA and proteins providesindependent information on direct contacts between components of theternary complex before and after heat shock. Various HSR1s (either invitro synthesized or purified from cell extracts) are 5′ [³²P] labeledwith T4 polynucleotide kinase. HSR1 footprinting with and withoutHSF/eEF1A is performed using FeEDTA-generated hydroxyl radicals thatattack the sugar moiety. OH• are particularly useful probes in this casesince, in contrast to base-modifying probes such as dimethyl sulfate, ornucleases, they do not discriminate between single- and double-strandedsites on the RNA (Celander, et al., Biochemistry 1990; 29:1355-1361).Fe(II)-EDTA-hydrogen peroxide solutions have been previously used by theinventors and co-workers to probe protein-RNA interactions intranscription complexes (Nudler, et al., Cell 1997; 89:33-41).

Hydroxyl radical footprinting is also used in relation to HSF1, todetermine which protein regions of HSF1 are in direct contact with othercomponents of the ternary complex. Specifically, an adaptation of thehydroxyl radical footprinting method of Heyduk and co-workers (Heyduk,et al., Biochemistry 1994; 33:9643-9650; Heyduk, et al., Proc. Natl.Acad. Sci. U.S.A. 1996; 93:10162-10166) is used as follows: 1) HSF1 is³³P-end labeled using an introduced recognition site for heart-muscleprotein kinase (HMPK); 2) in parallel reactions, HO•-mediated cleavageof labeled HSF1 itself and in the complex with eEF1A and/or HSR1 isperformed under single-hit conditions; 3) the cleavage products areanalyzed by denaturing PAGE and Phosphorlmager. Binding of the ligand(HSR1 and eEF1A) decreases polypeptide backbone solvent accessibility atresidues it contacts, protecting against HO•-cleavage, therefore,resulting in a gap in the ladder of cleavage products. The location ofthe ligand-binding site is read out directly from location of the gap inthe ladder.

The results obtained using the methods described above providecomprehensive information regarding the dynamic structural organizationof the HSR1/eEF1A/HSR1 ternary complex under stress and non-stressconditions. This information is useful for understanding the basicprincipals of HSF1 activation and also for designing the most effectivesmall antisense HSR1 oligonucleotides for in vivo inhibition of HSPsexpression (see Example 15, infra).

Example 9 Investigation of the Role of HSR1/eEF1A in Heat-Inducible HSF1Phosphorylation

As mentioned in the Background Section, the second step in the pathwayleading to transcription activation by HSF1 is its hyperphosphorylation(Christians et al., Crit. Care Med. 2002; 30(1 Supp):S43-S50). Sincehyperphosphorylation-dependant transcription activity of HSF1 trimersdepends on heat shock stimuli but not other stresses, e.g. salicylatetreatment (Jurivich, et al., J. Biol. Chem. 1995b; 270:24489-24495), itis hypothesized that HSR1/eEF1A not only induces HSF1 trimerization andDNA-binding but also regulates HSF1 phosphorylation upon heat shock. Itwas shown in previous studies that phosphorylation of Ser230 residue bycalcium/calmodulin dependent protein kinase II (CaMKII) as well asSer363 by c-Jun N-terminal kinase (JNK) activate transactivationfunction of HSF1 (Holmberg, et al., EMBO J. 2001; 20:3800-3810). In thepresent Example, the inventors set out to test whether HSR1/eEF1Apromotes HSF1 phosphorylation at Ser230, 263 upon heat shock byperforming in vitro HSF activation experiments in the presence of CaMKII(Promega) or JNK (Upstate Biotechnologies, Lake Placid, N. Y.) andradioactive ATP. HSF phosphorylation is performed in the presence orabsence of eEF1A and HSR1, at heat shock (43° C.) or normal (37° C.)temperature. The reaction is terminated and resolved on SDS-PAGE. Gelsare exposed to X-ray film to determine the extent of HSF1phosphorylation. HSF1 phosphorylation sites are mapped more preciselyusing methodology described in Example 8, supra.

Example 10 Investigation of Subcellular Localization of HSR1, eEF1A, andHSF Before and after Heat Shock

The subcellular distribution of the HSF1 activating components beforeand after heat shock is analyzed using conventional biochemical methodssuch as separation of nuclear extracts from that of cytoplasm andimmunofluorescent staining of fixed cells followed by observing withconfocal microscopy.

To test whether cytoplasm or nuclear extract of unstressed cells can beactivated in vitro by eEF1A and HSR1, cytoplasm and nuclear extracts areprepared as disclosed in Manalo, et al., Biochemistry 2002; 41:2580-2588and Manalo, et al., J. Biol. Chem. 2001; 276:23554-23561. Aliquots areincubated with eEF1A and in vitro transcribed HSR1 and subjected to EMSAanalysis.

The distribution of eEF1A between cytoplasm and nucleus during heatshock is analyzed in BHK and HeLa cells by immunostaining andimmunoprecipitation using monoclonal anti-eEF1A antibodies (UpstateBiotechnologies, Inc., Lake Placid, N. Y.). Cells are cultured oncoverslips (for immunstaining) or in flasks and subjected to heat shock.Then the cells are either fixed and stained with anti-eEF1A antibodiesor cytoplasmic and nuclear extracts are prepared from the cells andeEF1A is immunoprecipitated. Two different methods of protein fixationare used: paraformaldehyde (protein-protein and protein-nucleic acidcross-linking) and methanol (protein precipitation). Optimization of thestaining conditions is performed including duration of cell fixation,composition of the solutions used to permeabilize membranes and blockthe non-specific protein binding. A preliminary data obtained by theinventors indicate that there is heat shock dependent nuclearlocalization of a sub-population of eEF1A molecules. Curiously, eEF1Awas shown at least in one case to display nuclear localization inresponse to specific stimuli (Gangwani, et al., J Cell Biol 1998a;143:1471-1484).

To follow HSR1 subcellular localization in response to heat shock, theHSR1 antisense oligonucleotides that did not interfere with HSFactivation in reconstituted system are coupled with fluorescent dye andtransfected into BHK and HeLa cells. The cells then are either heatshocked or kept at normal growth temperature and observed usingfluorescent microscope.

Example 11 Investigation of HSF1/eEF1A/HSR1 Complex Formation with DNAIn Vivo by Chromatin Immunoprecipitation (Chip)

It is useful to understand what happens with the HSF1/eEF1A/HSR1 complexafter HSF activation and binding to DNA (HSE site) in vivo. To determinewhether eEF1A and/or HSR1 stay with HSF1 on the promoter and for howlong, chromatin immunoprecipitation (chIP) assay is used. The assaycombines reversible in vivo formaldehyde cross-linking withimmunoprecipitation and PCR. Protein-protein and protein-DNAcross-linking is induced in vivo on the heat shock inducible human HSP70promoter of the plasmid that is transiently transfected into HeLa cells.The cross-linked material is immunoprecipitated with antibodies to eEF1Aor HSF1. In a control experiment, eEF1A IP and HSF-1 IP from unstressedcells is performed. After reversing cross-linking and digestion ofproteins with proteinase K, the cross-linked DNA is isolated and thefragment of interest is amplified by quantitative real-time PCR. Next,the amount of PCR product from eEF1A IP is compared to that of HSF1. Thelatter is taken as 100% since under heat shock conditions most, if notall, heat shock promoters should be occupied by HSF1 trimers (Shopland,et al., Chromosoma 1996; 105:158-171). Real-time PCR allows comparisonof multiple reactions in their respective linear range, even if thesereactions follow very different kinetics. This can not be achieved by acommon PCR and quantifying their products on agarose gel, asconventionally performed in chIP. The above procedure is used toquantify the amount of HSR1 present in the precipitated cross-linkedcomplexes. In this case, single tube real-time RT-PCR is used. Thequantity of each of the components mentioned above is measured inunstressed cells, cells heat shocked (43° C.) for various periods oftime and cells that were heat shocked and allowed to recover at 37° C.in order to follow the kinetics of protein-protein and protein-RNAinteractions during HSF1 activation.

Example 12 Investigation of the Mechanism of HSF Activation in LowerEukaryotes (Drosophila and Yeast) and Other Members of HSF Family (HSF2,HSF4)

The power of genetic manipulation with Drosophila and yeast make thesespecies particular attractive in addressing fine molecular andphysiological details of the HSF activation process.

In Drosophila melanogaster, components of HSF activation system areidentified on polytene chromosomes. Polytene chromosomes have been usedextensively for direct visualization of various transcription andchromatin components (Simon, et al., Cell 1985; 40:805-817; Lis, et al.,Genes Dev. 2000; 14:792-803; Andrulis, et al., Genes Dev. 2000;14:2635-2649). Briefly, following heat shock, salivary gland isdissected, mounted on slide, and fixed by formaldehyde. The fixed slidesare then stained by incubation with commercial antibodies against HSFand eEF1A followed by several washes and incubation with appropriatesecondary fluorescent dye-conjugated (FITC and/or TRITC, JacksonImmunoresearch Laboratories, Inc.) antibody. Antibody is diluted in asolution containing 5% normal donkey serum to minimize non-specificbackground. Fluorescent microspheres are added to the samples stained bytwo antibodies to align color merges. DNA is visualized by staining withHoechst 33258 (Invitrogen, Calif.). Images are collected using eitherfluorescent or confocal microscope.

Currently, the general consensus holds that HSF in yeast isconstitutively trimeric and bound to HSE of heat shock promoters. Heatshock treatment is believed to induce transactivation function of HSFbut little or no additional DNA binding (Jakobsen, et al., Mol. Cell.Biol. 1988; 8:5040-5042 Jakobsen, et al., Mol. Cell. Biol. 1988;8:5040-5042; Sorger, et al., Nature 1987; 329:81-84). Yeast HSF alsodiffers from higher eukaryotic HSF1 in lacking the fourth leucine zipperdomain on its C-terminus. This domain has been implicated in inducibleHSF trimerization and acquisition of DNA binding activity (Bonner, etal., Mol. Biol. Cell 2000a; 11:1739-1751; Bonner, et al., J. Mol. Biol.2000b; 302:581-592). However, other data suggest that even in yeastexposure to elevated temperature can result in 10 to 20 fold increase inHSF binding to DNA (J. Lis, personal communication).

In the present Example, the methodology developed in mammalian andDrosophila systems is applied to address the mechanism of activation ofHSF in yeast. First, the existence of RNA similar to HSR1 in yeast cellsis verified. This is done by coupling HSF from Saccharomyces cerevisiaecells to activated Sepharose and incubating the resulting beads with thelysate of heat shocked cells followed by RNA extraction and sequencing(see Examples 1-3).

HSF2 is another member of HSF protein family (Mathew, et al., Mol. Cell.Biol. 2001; 21:7163-7171). Unlike HSF1, which is activated in responseto stress, HSF2 is activated during specific stages of development(Eriksson, et al., Int. J. Dev. Biol. 2000; 44:471-477; Min et al.,Biochim. Biophys. Acta. 2000; 1494:256-62; Loones, et al., Cell Mol.Life Sci. 1997; 53:179-190), in hemin-induced cell differentiation(Sistonen, et al., Mol. Cell Biol. 1992; 12:4104-4111), and in responseto inhibition of the ubiquitin-dependent proteosome (Mathew, et al.,Mol. Cell Biol. 1998; 18:5091-5098). HSF2 is an unstable protein whichexists in the cell in inactive monomeric and dimeric form but convertsto trimers upon activation (Wu, Ann. Rev. Cell Dev. Biol. 1995;11:441-469).

The most recently discovered member of HSF family, HSF4, is expressed intissue specific manner in two splice isoforms—HSF4a and HSF4b (Nakai,Chaperones. 1999; 4:86-93). The former lacks transactivation domainpresent in all HSF proteins and acts as a repressor of HSF1 mediatedtranscription (Zhang, et al., J. Cell Biochem. 2001; 82:692-703).

To determine whether HSF2 and HSF4 are also activated by anRNA-dependent mechanism similar to HSF1, these members of HSF family areexpressed in bacteria as either His6-tagged (SEQ ID NO: 56) orGST-fusion proteins, purified and covalently immobilized on Sepharose.The resulting HSF2- and HSF4-Sepharose is used to trapHSF2/HSF4-interacting proteins and RNA from the lysate of heat shockedor unstressed cells.

In summary, the experiments described in this section determine theintracellular compartment where the activation of HSF takes place andthe role of HSR1/eEF1A in HSF phosphorylation and transcriptionactivation. In addition, they allow to further characterize themechanism of interaction between HSR1 and HSF/eEF1A and possibly otherassociated factors in vivo and check the possibility that other HSFfamily members such as HSF2 and HSF4 require RNA for their activation.Finally, studies in Drosophila and yeast show the extent of evolutionaryconservation of the proposed mechanism of activation of heat shockresponse.

Example 13 Isolation of Cellular Factors that are Associated withHSF1-HSR1-eEF1a Complex Before and after Heat Shock

As mentioned in the Background Section, HSF1 is a target for manyfactors that are directly or indirectly involved in regulation of stressresponse. Proteins that have been shown to physically associate withHSF1 include several members of preinitiation transcription complex suchas mediator and activators (Park, et al., Mol. Cell 2001; 8:9-19; Mason,et al., J. Biol. Chem. 1997; 272:33227-33), transcription elongationfactor P-TEFb (Lis, et al., Genes Dev. 2000; 14:792-803), and SWI/SNFchromatin remodeling complex (Sullivan, et al., Mol. Cell Biol. 2001;21:5826-5837). Recruitment and/or activation of these factors byHSE-bound HSF1 is thought to play a critical role in the mechanism oftranscription activation of HSP genes. In addition, negative regulatorof HSF1 (HSBP1) has been shown to bind HSF1 directly to modulate itsactivity (Satyal et al., Genes Dev. 1998; 12:1962-74). HSF1 bindingproteins have been identified using either coimmunoprecipitation withantibodies against HSF1 or yeast two-hybrid screens with HSF1 domains asa bait.

To identify new HSF1-binding proteins and known proteins whoseinteraction with HSF1 is affected by the formation of theHSF1/HSR1/eEF1A ternary complex, in vivo crosslinking with formaldehyde(Hall, et al., J. Biol. Chem. 2002; 277:46043-46050) is combined withaffinity chromatography using biotinylated HSR1-antisenseoligonucleotides as a tag. Crosslinking (0.7% formaldehyde, 20 min, 37°C.) is performed in 10-20 flasks of cultured cells grown under stress(e.g., 1 h at 43° C.) and non-stress conditions. Proteins that arecrosslinked to the HSF1/HSR1/eEF1A complex are precipitated onNeutrAvidin™ beads (Pierce; >17 μg/ml of slurry beads) under strictconditions to remove non-specifically bound proteins (e.g., 2M KCl, milddetergents) using 3′-biotinylated and 5′-biotinylated HSR1 antisenseoligonucleotides. Several such oligonucleotides (e.g., 1^(HSR1),6^(HSR1), 14^(HSR1), see FIG. 5E) complementary to different parts ofHSR1 are used. A biotinylated oligonucleotide which is not complementaryto HSR1 is used as a negative control. After precipitation on beads andthorough washing, the crosslinking is reversed by boiling for 20 min.Precipitated proteins are resolved on SDS PAGE and silver-stained.MALDI-TOF analysis is used to identify the isolated proteins (Bar-Nahum,et al., Cell 2001; 106:443-451). Candidate proteins are cloned andanalyzed for their effect on HSF1 activation in vitro and HSP expressionin vivo (see Examples, supra).

Example 14 Design of Novel Anti-Cancer Drugs Based on HSR1 AntisenseOligonucleotides

HSPs (in particular HSP70) play a critical role in protecting the cellagainst apoptosis (see Background Section). As disclosed herein, thepresent inventors have identified a novel RNA, HSR1, that is involved inthe process of HSF1 activation in response to heat shock. As furtherdisclosed herein, the present inventors have demonstrated that HSR1antisense oligonucleotides are capable of inhibiting HSF1 activation invivo when transfected in BHK and HeLa cells and render them heatsensitive. This opens up an intriguing possibility of developing a novelanti-cancer agents based on HSR1 antisense oligonucleotides, which canbe used in conjunction with existing treatments to improve their effectby increasing the sensitivity of the cells to pro-apoptotic stimuli suchas thermo-, chemo-, and radiotherapeutic treatments.

Study of the Effect of HSR1 Antisense Oligonucleotides Transfection onCell Survival after Heat Shock

As disclosed in Example 4, supra, the present inventors have synthesizeda series of 45-mer oligonucleotides covering the entire HSR1 sequenceand screened them for the ability to suppress HSF1 activation in vitro.Four of these oligonucleotides effectively suppressed HSF1 activation inthe reconstituted system (FIG. 5E). Transfection of the most effectiveantisense oligonucleotide (6^(HSR1); SEQ ID NO: 12) into BHK and HeLacells had a strong inhibitory effect on HSF1 activation by heat shock invivo and rendered cells sensitive to heat (FIG. 6A). The data show thatthis treatment dramatically reduces the level of HSP expression and,therefore, promotes pro-apoptotic processes that are otherwise blockedby increased HSP expression.

To explore the possibility of using HSR1 antisense oligonucleotides totreat cancer, a model thermotolerant breast cancer cell line (Bcap37,maintained by the Cancer Institute of Zhejiang University; Wang, et al.,Biochem. Biophys. Res. Commun. 2002; 290:1454-1461) is transfected with6^(HSR1) oligonucleotide and the survival of cells is evaluated afterheat shock treatment (1 h at 43° C.) in comparison to that ofuntransfected cells. Cell survival plot is generated using flowcytometry analysis as well as Cell Titer Glo luminescent cell viabilityassay (Promega). The latter provides a method for determining the numberof viable cells in culture based on quantitation of ATP.

To confirm that HSR1 antisense oligonucleotide treatment indeed leads toreduction in HSP70 level in Bcap37 cells, lysates of heat shockedtransfected cells are analyzed by immunoblotting.

HSR1 antisense oligonucleotides which show the strongest effect on cellsurvival and HSP expression may be further optimized (e.g., byincreasing their resistance to nucleases, increasing the efficiency oftheir targeting to cells, increasing their sequence specificity [e.g.,by introducing phosphothioate or morpholino modifications or using LNA],and reducing the size) making them even more potent in inhibition ofcell survival and inhibition of HSP expression.

Study of the Effect of HSR1 Antisense Oligonucleotides on TumorProgression in Nude Mice

The HSR1 antisense oligonucleotides selected as the most potent in cellculture assays in the previous section are used to develop a product,which, when delivered directly to the tumor, reduces expression of HSPsin cancer cells thereby making them more susceptible to apoptoticstimuli and cytotoxic agents. Specifically, nude mice (BALB/c nu/nustrain [Taconic]) are inoculated by subcutaneous and bilateral injectionwith thermotolerant breast cancer Bcap37 cells to induce tumorformation. When the tumors reach 3-4 mm in diameter, the mice areadministered phosphothioate-modified anti-HSR1 oligonucleotides byinjection into tumors, allowed 16-24 h for the oligonucleotide uptake,and heat shocked for 30 min at 43° C. in an incubator. As previouslymentioned, phosphothioate or morpholino modification of theoligonucleotides is required to minimize their susceptibility toexonucleases and ensure adequate stability in the tissue. In addition toheat-shock, a separate group of experimental mice with anti-HSR1oligonucleotide pretreated Bcap37 tumors are treated with a standardanti-cancer drug (e.g., fluorouridine). In all cases, the treatment iscontinued for one week and the tumor size is assessed by measuring itsdimensions and calculating volume. Three control groups of animalsreceive heat shock treatment without antisense oligonucleotides, witholigonucleotides of the same length but randomized sequence, and notreatment at all. Similar methodology have been used recently to showthat blocking of HSF1 by dominant-negative mutant leads to enhancedefficiency of heat shock treatment of tumor induced in nude mice byinjection of Bcap37 cells (Wang, et al., Biochem. Biophys. Res. Commun.2002; 290:1454-1461).

In parallel, tumor tissues from treated and control animals are excised,homogenized and assayed for the activation of HSF1 by EMSA. The level ofHSP72 production is determined as well using standard immunoblottingtechniques.

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Numerous references, including patents, patent applications and variouspublications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed here. All references cited and/or discussed in thisspecification (including references, e.g., to biological sequences orstructures in the GenBank, PDB or other public databases) areincorporated herein by reference in their entirety and to the sameextent as if each reference was individually incorporated by reference.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

1. An isolated constitutively active fragment of a eukaryotic Heat ShockRNA (HSR1) lacking from 100 to 150 3′-terminal HSR1 nucleotides, whichfragment has at least 95% sequence identity to at least 100 consecutivenucleotides of SEQ ID NO:
 35. 2. The constitutively active HSR1 fragmentof claim 1 consisting of SEQ ID NO:
 35. 3. A recombinant vector encodingthe constitutively active HSR1 fragment of claim
 1. 4. An isolatedpolynucleotide molecule encoding the constitutively active HSR1 fragmentof claim
 1. 5. A host cell which has been transformed or transfectedwith the recombinant vector of claim 3.